Ginkgo biloba extract protects human neuroblastoma SH-SY5Y cells against oxidative glutamate toxicity by activating redoxosome-p66Shc

Abstract

Ginkgo biloba extract (GBE), a traditional Chinese herbal medicine component, is widely used to alleviate symptoms of neurodegenerative diseases. It has been confirmed that GBE exerts its pharmacological effect mainly due to its antioxidant activity; however, the molecular mechanism responsible for this effect remains unclear. The aim of the present study was to investigate the detailed mechanism of GBE, the main component of Gingko biloba dropping medicine, against oxidative glutamate toxicity in human neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were untreated or pretreated with GBE followed by glutamate stimulation. Cell viability was assessed using an MTT assay. In addition, oxidative stress indexes, including intracellular ROS generation and NADPH oxidase and caspase activity, were also measured. The protein expression of key signaling factors involved in the redoxosome-p66Shc pathway was evaluated to elucidate the neuroprotective effect of GBE. The results showed that GBE treatment significantly attenuated the glutamate-induced cytotoxicity in SH-SY5Y cells by suppressing oxidative stress. A mechanical study revealed that redoxosome-p66Shc activation was associated with glutamate-induced cytotoxicity, which caused mitochondrial dysfunction and cell death. Interestingly, GBE treatment attenuated the activation of redoxosome-p66Shc in a dose-dependent manner, which suggested that the protective effect of GBE on SH-SY5Y cells against oxidative glutamate toxicity may be mediated by the modulation of redoxosome-p66Shc signaling. The current findings contribute to a better understanding of the therapeutic effect of GBE and indicate that redoxosome-p66Shc signaling might be a novel therapeutic target in the prevention and/or treatment of neurodegenerative diseases.

Keywords: Ginkgo biloba extract; glutamate; neurodegenerative diseases; redoxosome-p66Shc signaling.

Figure 1

Protective effect of GBE against Glu-induced cytotoxicity in SH-SY5Y cells. (A) Cells were treated with various concentrations of GBE (0, 25, 50, 100 µg/ml) for 24 h and cell viability was evaluated using an MTT assay. (B) Cells were pretreated with various concentrations of GBE (0, 25, 50, 100 µg/ml) for 24 h and then exposed to Glu (50 mM) for 6 h. After incubation, cell viability was evaluated using an MTT assay. ^**P<0.01 vs. control; ^#P<0.05 and ^##P<0.01 vs. Glu alone. Glu, glutamate; GBE, Ginkgo biloba extract.

Figure 2

Protective effect of GBE against Glu-induced oxidative injury in SH-SY5Y cells. Cells were pre-treated with various concentrations of GBE (0, 25, 50, 100 µg/ml) for 24 h and then exposed to Glu (50 mM) for 2 h. (A) ROS level was evaluated using CM-H2DCFDA. (B) H₂O₂ level was evaluated using an Amplex Red assay. ^**P<0.01 vs. control; ^##P<0.01 vs. Glu alone. Glu, glutamate; GBE, Ginkgo biloba extract; ROS, reactive oxygen species.

Figure 3

Protective effect of GBE against Glu-induced redoxosome activation in SH-SY5Y cells. Cells were pretreated with various concentrations of GBE (0, 25, 50, 100 µg/ml) for 24 h and then exposed to glutamate (50 mM) for 2 h. (A) Protein levels of p-SRC, SRC, p-Vav2 and Vav2 were evaluated by western blot analysis. (B) Representative Rac1 western blot is shown. (C) NADPH oxidase activity was measured. ^**P<0.01 vs. control; ^##P<0.01 vs. Glu alone. Glu, glutamate; GBE, Ginkgo biloba extract; p, phosphorylated; SRC, proto-oncogene tyrosine-protein kinase Src; Vav2, guanine nucleotide exchange factor VAV2.

Figure 4

Effect of redoxosome inhibitors against glutamate-induced oxidative stress in SH-SY5Y cells. Cells were pretreated with Rac inhibitor N (80 µM) or NADPH oxidase inhibitor V (10 µM) for 6 h and then exposed to Glu (50 mM) for 2 h. (A) ROS level was evaluated using CM-H2DCFDA. (B) H₂O₂ level was evaluated using an Amplex Red assay. ^**P<0.01 vs. control; ^##P<0.01 vs. Glu alone. N, NSC23766; V, VAS2870; Glu, glutamate; ROS, reactive oxygen species.

Figure 5

Role of p66Shc in glutamate-induced redoxosome activation in SH-SY5Y cells. Cells were pretreated with Rac inhibitor NSC23766 (80 µM) or NADPH oxidase inhibitor VAS2870 (10 µM) for 6 h and then exposed to Glu (50 mM) for 2 h. The protein levels and distribution of p-p66Shc and p66Shc were evaluated by western blot analysis. ^**P<0.01 vs. control; ^##P<0.01 vs. Glu alone. N, NSC23766; V, VAS2870; p, phosphorylated; Glu, glutamate; Mito, mitochondrial fraction.

Figure 6

Protective effect of GBE against Glu-induced p66Shc activation in SH-SY5Y cells. Cells were pretreated with various concentrations of GBE (0, 25, 50, 100 µg/ml) for 24 h and then exposed to Glu (50 mM) for 2 h. The protein levels and distribution of p-p66Shc and p66Shc in the (A) cytosolic fraction and (B) mitochondrial fraction were evaluated by western blot analysis. ^**P<0.01 vs. control; ^##P<0.01 vs. Glu alone. Glu, glutamate; GBE, Ginkgo biloba extract; Mito, mitochondrial fraction; p, phosphorylated.

Figure 7

Protective effect of GBE against glutamate-induced mitochondrial function in SH-SY5Y cells. Cells were pretreated with various concentrations of GBE (0, 25, 50, 100 µg/ml) for 24 h and then exposed to Glu (50 mM) for 6 h. (A) MFI of Rh123. (B) Caspase-3 activity. (C) Mito Cyto c and Cyto Cyto c levels were determined by western blot analysis. ^**P<0.01 vs. control; ^##P<0.01 vs. Glu alone. Glu, glutamate; GBE, Ginkgo biloba extract; MFI, mean fluorescence intensity; Mito Cyto c, mitochondrial cytochrome c; Cyto Cyto c, cytosolic cytochrome c.

Figure 8

Effect of p66shc knockdown on Glu-induced mitochondrial function in SH-SY5Y cells. Cells with or without p66Shc knockdown were exposed to Glu (50 mM) for 6 h. (A) p66shc knockdown was assessed by western blot analysis. ^**P<0.01 vs. scrambled siRNA. (B) MFI of Rh123. (C) Caspase-3 activity. (D) Mito Cyto c and Cyto Cyto c levels were determined by western blot analysis. ^**P<0.01 vs. CT; ^##P<0.01 vs. CT with Glu treatment. Glu, glutamate; GBE, Ginkgo biloba extract; MFI, mean fluorescence intensity; Mito Cyto c, mitochondrial cytochrome c; Cyto Cyto c, cytosolic cytochrome c; KD, knockdown; siRNA, small interfering RNA.

Figure 9

Proposed molecular mechanism underlying the neuroprotective effect of GBE against glutamate-induced oxidative toxicity in SH-SY5Y cells. GBE, Ginkgo biloba extract; SRC, proto-oncogene tyrosine-protein kinase Src; Vav2, guanine nucleotide exchange factor VAV2; P, phosphorylated; NOX, NADPH oxidase.