Edible bird's nest ameliorates oxidative stress-induced apoptosis in SH-SY5Y human neuroblastoma cells

Abstract

Background: Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting the senile population with manifestation of motor disability and cognitive impairment. Reactive oxygen species (ROS) is implicated in the progression of oxidative stress-related apoptosis and cell death of the midbrain dopaminergic neurons. Its interplay with mitochondrial functionality constitutes an important aspect of neuronal survival in the perspective of PD. Edible bird's nest (EBN) is an animal-derived natural food product made of saliva secreted by swiftlets from the Aerodamus genus. It contains bioactive compounds which might confer neuroprotective effects to the neurons. Hence this study aims to investigate the neuroprotective effect of EBN extracts in the neurotoxin-induced in vitro PD model.

Methods: EBN was first prepared into pancreatin-digested crude extract and water extract. In vitro PD model was generated by exposing SH-SY5Y cells to neurotoxin 6-hydroxydopamine (6-OHDA). Cytotoxicity of the extracts on SH-SY5Y cells was tested using MTT assay. Then, microscopic morphological and nuclear examination, cell viability test and ROS assay were performed to assess the protective effect of EBN extracts against 6-OHDA-induced cellular injury. Apoptotic event was later analysed with Annexin V-propidium iodide flow cytometry. To understand whether the mechanism underlying the neuroprotective effect of EBN was mediated via mitochondrial or caspase-dependent pathway, mitochondrial membrane potential (MMP) measurement and caspase-3 quantification were carried out.

Results: Cytotoxicity results showed that crude EBN extract did not cause SH-SY5Y cell death at concentrations up to 75 μg/ml while the maximum non-toxic dose (MNTD) of water extract was double of that of crude extract. Morphological observation and nuclear staining suggested that EBN treatment reduced the level of 6-OHDA-induced apoptotic changes in SH-SY5Y cells. MTT study further confirmed that cell viability was better improved with crude EBN extract. However, water extract exhibited higher efficacy in ameliorating ROS build up, early apoptotic membrane phosphatidylserine externalization as well as inhibition of caspase-3 cleavage. None of the EBN treatment had any effect on MMP.

Conclusions: Current findings suggest that EBN extracts might confer neuroprotective effect against 6-OHDA-induced degeneration of dopaminergic neurons, particularly through inhibition of apoptosis. Thus EBN may be a viable nutraceutical option to protect against oxidative stress-related neurodegenerative disorders such as PD.

Figure 1

Cytotoxic effect of EBN extracts on SH-SY5Y cells. Cytotoxicity percentage of SH-SY5Y cells upon 48 hours treatment with either S1 or S2 was tested across a wide range of concentration from 0 to 500 μg/ml and MTT assay was carried out. Maximum non-toxic dose was then determined from the graph. The data shown are means ± S.D. of three independent experiments performed in triplicates.

Figure 2

Effect of EBN extracts on morphological and nuclear changes of 6-OHDA-challenged SH-SY5Y cells. Microscopic images were taken after 48 hours of treatment. Figures A-D are bright field images while Figures E-H are fluorescent images taken after Hoechst 33258 staining. Figures A and E: control group; Figures B and F: 6-OHDA group; Figures C and G: S1 MNTD + 6-OHDA-treated group; Figures D and H: S2 MNTD + 6-OHDA-treated group. Cell shrinkage is indicated by cell losing its distinctive neuronal shape and has becomes smaller in size (yellow arrow in Figure 2B), DNA fragmentation is indicated by cluster of nuclei fragments (red arrow in Figure 2F), shrunken cell is indicated by smaller and distorted nuclei (green arrow in Figure 2F) while nuclear chromatin condensation is indicated by brightly fluorescent nuclei (white arrow in Figure 2F).

Figure 3

Effect of EBN extracts on 6-OHDA-challenged SH-SY5Y cell viability. Cell viability was assessed with MTT assay and data shown are means ± S.D. of three independent experiments performed in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001 versus untreated control cells while ^#P < 0.05, ^##P < 0.01; ^###P < 0.001 versus 6-OHDA treated cells.

Figure 4

Effect of EBN extracts on intracellular reactive oxygen species (ROS) production in 6-OHDA-challenged SH-SY5Y cells. Intracellular ROS levels of treated groups were assessed with DCFH-DA assay and data shown are means ± S.D. of three independent experiments performed in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001 versus untreated control cells while ^#P < 0.05, ^##P < 0.01; ^###P < 0.001 versus 6-OHDA treated cells.

Figure 5

Effect of EBN extracts on 6-OHDA-induced apoptosis in SH-SY5Y cells. Cells treated with EBN extracts for 48 hours were analyzed by Annexin V-propidium iodide double staining. Representative plots of propidium iodide versus Annexin V-FITC fluorescence signals are shown (A-J). Results shown are the mean ± S.D. of three independent experiments. *P < 0.05; **P < 0.01 versus untreated control cells while ^#P < 0.05, ^##P < 0.01; ^###P < 0.001 versus 6-OHDA treated cells.

Figure 6

Effect of EBN extracts on mitochondrial membrane potential (MMP) in 6-OHDA-challenged SH-SY5Y cells. MMP was assessed with mitochondria-selective JC-1 dye and results shown are the mean ± S.D. for three independent experiments. *P < 0.05; **P < 0.01 versus untreated control cells while ^#P < 0.05 versus 6-OHDA treated cells.

Figure 7

Effect of EBN extracts on cleavage of caspase-3 in SH-SY5Y cells challenged with 6-OHDA. The release of active caspase-3 into cytosol was assessed by immunostaining using FITC-conjugated antibody and results were generated from flow cytometry. Results shown are the mean ± S.D. of three independent experiments. **P < 0.05; **P < 0.01 versus untreated control cells while ^#P < 0.05, ^##P < 0.01versus 6-OHDA treated cells.