Protective Effect of Amber Extract on Human Dopaminergic Cells against 6-Hydroxydopamine-Induced Neurotoxicity

Abstract

Parkinson's disease (PD) is the second most common progressive neurodegenerative disease, after Alzheimer's disease. In our previous study, we found that amber-a fossilized plant resin-can protect cells from apoptosis by decreasing the generation of reactive oxygen species (ROS). In this study, we focused on the effect of amber on 6-hydroxydopamine-induced cell apoptosis in the human neuroblastoma cell line SHSY5Y (one model for PD). Initially, we determined the protective effect of amber on the PD model. We found that amber extract has a protective effect against 6-hydroxydopamine-induced cell apoptosis. The decrease in ROS, cleaved caspase-3, pERK, and extracellular signal-regulated kinase (ERK) protein levels confirmed that amber extract decreases apoptosis via the ROS-mediated ERK signaling pathway. Furthermore, we determined the effects of amber extract on autophagy. The results showed that amber extract increased the levels of LC3II and Beclin-1, suggesting that amber extract can protect neuronal cells against 6-hydroxydopamine-induced cell apoptosis by promoting autophagy.

Keywords: 6-hydroxydopamine; amber; autophagy; extracellular signal-regulated kinase pathway.

Figure 1

Effect of amber extract on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity: SH-SY5Y cells were cultured in DMEM/F12 medium with 10% FBS. After 24 h of seeding, the cells were pretreated with amber extract for 24 h, and then the medium was replaced with fresh 6OHDA-containing medium for another 24 h, after which cell viability was measured using the MTT assay. Control (Ct): (6-OHDA/amber) −/−, 6-OHDA: (6-OHDA/amber) +/−, Amber: (6-OHDA/amber) (+/15 µg/mL; +/25 µg/mL, +/50 µg/mL). (A) Cytotoxicity of amber extract. (B) Effect of amber extract on 6-OHDA-induced cell death. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide assay. The results are expressed as the mean ± standard deviation. n ≥ 3, * p < 0.05 vs. Control, ** p < 0.01 vs. Control, ## p < 0.01 vs. 6-OHDA.

Figure 2

Effect of amber extract on 6-hydroxydopamine (6-OHDA)-induced cell apoptosis: SH-SY5Y cells were cultured in DMEM/F12 medium with 10% FBS. After 24 h of seeding, the cells were pretreated with amber extract for 24 h, and then the medium was replaced with fresh 6OHDA-containing medium for another 6 h for apoptosis assay, and 24 h for WB. Control (Ct): (6-OHDA/amber) −/−, 6-OHDA: (6-OHDA/amber) +/−, Amber: (6-OHDA/amber) (+/15 µg/mL; +/25 µg/mL, +/50 µg/mL). (A) Apoptosis analyzed using a fluorescence microplate reader. (B) Representative Western blot image of caspase-3 and cleaved caspase-3. (C) The protein bands of cleaved caspase-3 were quantified using ImageJ. The results are expressed as the mean ± standard deviation. n ≥ 3, ** p < 0.01 vs. Control, # p < 0.05 vs. 6-OHDA, ## p < 0.01 vs. 6-OHDA.

Figure 3

Effect of amber extract on 6-hydroxydopamine (6-OHDA)-induced intracellular reactive oxygen species (ROS) generation: SH-SY5Y cells were cultured in DMEM/F12 medium with 10% FBS. After 24 h of seeding, the cells were pretreated with amber extract for 24 h, and then the medium was replaced with fresh 6OHDA-containing medium for another 6 h for ROS assay, and 24 h for WB. Control (Ct): (6-OHDA/amber) −/−, 6-OHDA: (6-OHDA/amber) +/−, Amber: (6-OHDA/amber) (+/15 µg/mL; +/25 µg/mL, +/50 µg/mL) (A) ROS analyzed using a fluorescence microplate reader. (B) Representative Western blot image of pERK and extracellular signal-regulated kinase. (C) The protein bands were quantified using ImageJ. The results are expressed as the mean ± standard deviation. n ≥ 3 * p < 0.05 vs. Control, ** p < 0.01 vs. Control, # p < 0.05 vs. 6-OHDA.

Figure 4

Effect of amber extract on autophagy-related gene expression: SH-SY5Y cells were cultured in DMEM/F12 medium with 10% FBS. After 24 h of seeding, the cells were pretreated with amber extract for 24 h, and then the medium was replaced with fresh 6OHDA-containing medium for another 24 h. Control (Ct): (6-OHDA/amber) −/−, 6-OHDA: (6-OHDA/amber) +/−, Amber: (6-OHDA/amber) (+/15 µg/mL; +/25 µg/mL, +/50 µg/mL) (A) Representative Western blot image of Beclin-1 and LC3. (B) The protein bands of Beclin-1 were quantified using ImageJ. (C) The protein bands of LC3 Ⅱ were quantified using ImageJ. (D) The relative ratios of LC3 II/LC3 I bands’ density. The results are expressed as the mean ± standard deviation. n ≥ 3 * p < 0.05 vs. Control, # p < 0.05 vs. 6-OHDA, ## p < 0.01 vs. 6-OHDA.