Baicalin Attenuated A β 1-42-Induced Apoptosis in SH-SY5Y Cells by Inhibiting the Ras-ERK Signaling Pathway

Abstract

Alzheimer's disease (AD) is a serious neurodegenerative disease. It is widely believed that the accumulation of amyloid beta (Aβ) in neurons around neurofibrillary plaques is the main pathological characteristic of AD; however, the molecular mechanism underlying these pathological changes is not clear. Baicalin is a flavonoid extracted from the dry root of Scutellaria baicalensis Georgi. Studies have shown that baicalin exerts excellent anti-inflammatory and neuroprotective effects. In this study, an AD cell model was established by exposing SH-SY5Y cells to Aβ _1-42 and treating them with baicalin. Cell survival, cell cycle progression, and apoptosis were measured by MTT, flow cytometry, and immunofluorescence assays, respectively. The expression levels of Ras, ERK/ERK phosphorylation (p-ERK), and cyclin D1 were measured by Western blotting. In addition, whether the MEK activator could reverse the regulatory effect of baicalin on Ras-ERK signaling was investigated using Western blotting. We found that baicalin improved the survival, promoted the proliferation, and inhibited the apoptosis of SH-SY5Y cells after Aβ _1-42 treatment. Baicalin also ameliorated Aβ _1-42-induced cell cycle arrest at the S phase and induced apoptosis. Furthermore, baicalin inhibited the levels of Ras, p-ERK, and cyclin D1 induced by Aβ, and this effect could be reversed by the MEK activator. Therefore, we suggest that baicalin may regulate neuronal cell cycle progression and apoptosis in Aβ _1-42-treated SH-SY5Y cells by inhibiting the Ras-ERK signaling pathway. This study suggested that baicalin might be a useful therapeutic agent for senile dementia, especially AD.

Figure 1

Baicalin improved the survival of Aβ_1-42-treated SH-SY5Y cells. (a) Cell viability of SH-SY5Y cells treated with 10 μM Aβ_1-42 for different times. (b) Cell viability of SH-SY5Y cells treated with different concentrations of baicalin. (c) Cell viability of Aβ_1-42-treated SH-SY5Y cells treated with different concentrations of baicalin. (d) LDH of Aβ_1-42-treated SH-SY5Y cells treated with different concentrations of baicalin. The levels of cell viability and LDH are presented as the mean ± SD. N = 6, ∗ represents P < 0.05, and ∗∗ represents P < 0.01.

Figure 2

Effect of baicalin on Aβ_1-42-treated SH-SY5Y cell apoptosis. (a) Control group. (b) Model group (10 μM Aβ_1-42). (c) 5 μM baicalin treatment group. (d) 10 μM baicalin treatment group. (e) 20 μM baicalin treatment group. (f) Apoptosis rate. The apoptosis rate is presented as the mean ± SD. N = 5; ∗∗ represents P < 0.01.

Figure 3

Aβ_1-42-induced apoptosis in SH-SY5Y cells was inhibited by baicalin treatment. SH-SY5Y cells were treated with 5 μM, 10 μM, and 20 μM baicalin for 24 h and analyzed by TUNEL fluorescence staining with a NIKON A1⁺ confocal microscope. The experiment was repeated twice. A total of 100 DAPI-positive nuclei were counted from three separate fields, and the percentage of apoptosis was calculated based on the number of TUNEL-positive nuclei in each field. The TUNEL-positive rate is presented as the mean ± SD. N = 5; ∗ represents P < 0.05.

Figure 4

Effect of baicalin on the cell cycle of Aβ_1-42-treated SH-SY5Y cells. (a) Control group. (b) Model group (10 μM Aβ_1-42). (c) 5 μM baicalin treatment group. (d) 10 μM baicalin treatment group. (e) 20 μM baicalin treatment group. (f) The data are presented as the mean ± SD. N = 5; compared with the model group, ## represents P < 0.01; compared with the model group, ∗∗ represents P < 0.01.

Figure 5

Inhibition of Ras-ERK signaling pathway activation in Aβ_1-42-treated SH-SY5Y cells by baicalin. (a) SH-SY5Y cell extracts were prepared and analyzed by Western blotting using Ras, P-ERK, total ERK, and cyclin D1 antibodies. β-Actin antibodies were used as a reference. (b) Ras protein expression normalized to β-actin. (c) p-ERK levels normalized to total ERK expression. (d) Cyclin D1 expression normalized to β-actin. The protein expression is presented as the mean ± SD. N = 3, ∗ represents P < 0.05, and ∗∗ represents P < 0.01.

Figure 6

Baicalin inhibited Ras-ERK signaling and cyclin D1 expression in Aβ_1-42-treated SH-SY5Y cells, and this effect was reversed with a MEK activator. (a) SH-SY5Y cell extracts were prepared and analyzed by Western blotting using P-ERK, total ERK, and cyclin D1 antibodies. β-Actin antibodies were used as a reference. (b) p-ERK levels normalized to total ERK expression; cyclin D1 expression normalized to β-actin. The protein expression is presented as the mean ± SD. N = 3, ∗ represents P < 0.05, and ∗∗ represents P < 0.01.