Neuroprotective effect of phospholipase A2 from Malaysian Naja sumatrana venom against H2O2-induced cell damage and apoptosis

Abstract

Oxidative stress is one of the factors involved in the pathogenesis of several neurodegenerative diseases. It has been reported that a secretory phospholipase A₂ known as A2-EPTX-NSm1a has lower cytotoxicity in neuronal cells compared to its crude Naja sumatrana venom. In this study, A2-EPTX-NSm1a was tested for its neuroprotective activity on human neuroblastoma cells (SH-SY5Y) differentiated into cholinergic neurons against oxidative stress induced by hydrogen peroxide (H₂O₂). H₂O₂ treatment alone increased the caspase-3 and caspase-8 activities, whereas pre-treatment with A2-EPTX-NSm1a reduced the activity of these apoptosis-associated proteins. Moreover, A2-EPTX-NSm1a protects the morphology and ultrastructure of differentiated SH-SY5Y cells in the presence of H₂O₂. Oxidative stress increased the number of small mitochondria. Further evaluation showed the size of mitochondria with a length below 0.25 µm in oxidative stress conditions is higher than the control group, suggesting mitochondria fragmentation. Pre-treatment with A2-EPTX-NSm1a attenuated the number of mitochondria in cells with H₂O₂ Furthermore, A2-EPTX-NSm1a altered the expression of several neuroprotein biomarkers of GDNF, IL-8, MCP-1, TIMP-1, and TNF-R1 in cells under oxidative stress induced by H₂O₂. These findings indicate that anti-apoptosis with mitochondria-related protection, anti-inflammatory effect, and promote expression of important markers for cell survival may underlie the neuroprotective effect of A2-EPTX-NSm1a in cholinergic rich human cells under oxidative stress, a vital role in the neuronal disorder.

Keywords: apoptosis; inflammation; mitochondria; neurodegenerative disease; neuroprotection; snake venom phospholipase A2.

FIGURE 1

Differentiation of SH-SY5Y cells. (A) SH-SY5Y were differentiated as in the line chart. (B) Phase contrast of SH-SY5Y cells morphology on days 0, 4 and 8. Neurite extension labelled in red arrow. Scale bar = 200 μM. (C) Differentiation of SH-SY5Y showed modification in the cell number, (D) neurite length and (E) AChE activity. Data were presented as means ± SD, where p < 0.05 is considered significant. **** indicated p < 0.001.

FIGURE 2

Cell proliferation and neuroprotection of a secretory phospholipase A2, A2-EPTX-NSm1a in differentiated SH-SY5Y cell lines. (A) Cytotoxicity of H₂O₂ on differentiated SH-SY5Y cells. (B) Cell proliferation of A2-EPTXNSm1a was evaluated at different times of incubation. (C) Neuroprotection assay of A2-EPTX-NSm1a. (D) Phase contrast of differentiated SH-SY5Y in the different groups - untreated (control), H₂O₂ only (oxidative group), pre-treatment of 0.977 µg/ml A2-EPTX-NSm1a followed by H₂O₂ and 0.977 µg/ml A2-EPTX-NSm1a only after 24 h of incubation time. Scale bar = 200 μM.

FIGURE 3

Effect of pre-challenged A2-EPTX-NSm1a on caspase-3 and caspase-8 activity in differentiated SH-SY5Y with H₂O₂. (A) Relative Caspase-3 activity (fold change); (B) Relative Caspase-8 activity (fold change) All experiment was conducted in triplicate (n = 3). Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 as compared to the H₂O₂-treated group.

FIGURE 4

Representative cell structure images of differentiated SH-SY5Y observed in an electron microscope. Image of differentiated SH-SY5Y cells in (A) untreated group (Control), (B) H₂O₂-induced neurotoxicity group, (C) addition of pre-treatment of A2-EPTX-NSm1a following the addition of H₂O₂ and (D) A2-EPTX-NSm1a alone at 24 h of incubation. Scale bar = 1 μm. Arrow showed the formation of membrane bubbling due to cell apoptosis induced by H₂O₂.

FIGURE 5

Observation of mitochondrial morphology from electron microscope. (A) Morphology of mitochondria in each group observed after 24 h of treatment in a representative of cell. (B) Number of mitochondria in the area has been quantified and the length of mitochondria in each group were measured using ImageJ analysis. Scale bar = 200 nm. Data showed mean ± SEM and marked with ** indicating p < 0.01, *** as p < 0.005, and **** marked as p < 0.001.

FIGURE 6

A2-EPTX-NSma1 effects on the neuro-markers expression levels in the differentiated SH-SY5Y with and without oxidative stress conditions. The expression level of proteins was analysed using normalized signals extracted from Agilent Feature Extraction software. Values are collected from quadruplicates of a specific protein and expressed as mean ± standard deviation (n = 3). Protein expression is considered as significant as compared to the H₂O₂ group with p < 0.05 based on statistical analysis using one-way ANOVA followed by Tukey’s post-test. ns as significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001.