A secretory phospholipase A2-mediated neuroprotection and anti-apoptosis

Abstract

Background: Phospholipase A2 liberates free fatty acids and lysophospholipids upon hydrolysis of phospholipids and these products are often associated with detrimental effects such as inflammation and cerebral ischemia. The neuroprotective effect of neutral phospholipase from snake venom has been investigated.

Results: A neutral anticoagulant secretory phospholipase A2 (nPLA) from the venom of Naja sputatrix (Malayan spitting cobra) has been found to reduce infarct volume in rats subjected to focal transient cerebral ischemia and to alleviate the neuronal damage in organotypic hippocampal slices subjected to oxygen-glucose deprivation (OGD). Real-time PCR based gene expression analysis showed that anti-apoptotic and pro-survival genes have been up-regulated in both in vivo and in vitro models. Staurosporine or OGD mediated apoptotic cell death in astrocytoma cells has also been found to be reduced by nPLA with a corresponding reduction in caspase 3 activity.

Conclusion: We have found that a secretory phospholipase (nPLA) purified from snake venom could reduce infarct volume in rodent stroke model. nPLA, has also been found to reduce neuronal cell death, apoptosis and promote cell survival in vitro ischemic conditions. In all conditions, the protective effects could be seen at sub-lethal concentrations of the protein.

Figure 1

Histological analysis of brain sections. (a) TTC stained coronal brain sections (2 mm thick) of rats treated with 0.15 μg/g body weight nPLA intravenously at 0 min, 5 min and 15 min post-occlusion (n = 6). Surviving cells stained red whilst dead cells remained white. (b) Infarct volumes are expressed as a percentage of the vehicle control ± SEM. *, p < 0.05; **, p < 0.01 using single-factor ANOVA followed by Dunnett multiple comparison test.

Figure 2

Anti-apoptotic effect of nPLA (a) Histological and TUNEL analysis of brain sections. High power (×120) photomicrographs of haematoxylin and eosin-stained (b) TUNEL-stained striatal and cortical tissue. Nuclei are indicated by arrows. Rats subjected to transient MCAo were injected intravenously with either vehicle or 0.15 μg/g body weight nPLA. Sham-operated animals underwent the entire surgical procedure other than suture insertion. (c) Quantitative gene expression (real-time PCR) analysis. Gene expression analysis on selected genes for sham operated (Sham), MCAo and MCAo+nPLA brain samples. All measurements were performed in triplicate. Values are expressed as fold change ± SEM, * p < 0.05; ** p < 0.01 by unpaired Student's t-test.

Figure 3

(a) Effect of nPLA (0.01 μM) on dose-dependent STS-induced cell death in astrocytoma cells (CRL1718™). Cell viability assay was carried out following 16 hrs exposure to various concentrations of STS or STS + 0.01 μM nPLA. Asterisks denote difference from its each individual STS concentration treatment. Values are expressed as percentage of viability (to the respective treatment without nPLA) ± SEM, * p < 0.05 by unpaired Student's t-test (n = 9). (b) Effect of nPLA on STS-induced cell death by caspase-3 activity assay. Control: no reagents, STS: 1.5 μM STS, STS + nPLA: 1.5 μM STS+0.01 μM nPLA. Data shown are mean ± SEM (n = 18). (c) Effect of nPLA on CRL1718™ astrocytoma cells subjected to OGD. Cells were subjected to 2 hr OGD and nPLA (0.038 μM or 0.01 μM) was added in the culture during the ischemic period. Cell viability assay was carried out following a 24 hr of reperfusion. Caspase-3 assay was carried out on the respective cell lysates. Data shown are mean ± SEM (n = 3, conducted in triplicates, p < 0.05, two tail Student's t test).

Figure 4

Organotypic hippocampal culture subjected to OGD. (a) Slices subjected to oxygen-glucose deprived (OGD) condition in the presence of nPLA (0.037 μM-0.15 μM) and propidium iodide (5 μg/ml). Confocal microscopy images show that brighter areas correspond to a high propidium iodide [24] uptake level indicating a higher density of dead or damaged cells. (b) Percentage of cell survival, calculated based on PI intensity on each CA1 and CA3 regions. Each point represents the mean ± SEM (n = 8). #; p < 0.05 and *; p < 0.01. (c) Quantitation of gene expression using real-time PCR. Statistical analysis was performed using one-way analyses of variance (ANOVA; *; p ≤ 0.005.) and a t-test to determine significance between groups for LDH assay (n = 6) and real-time PCR (n = 9).

Figure 5

Microarray analysis on the endogenous phospholipases. (a) Hierachical clustering of all the phospholipases detected in our global gene expression studies. Signal log(2) ratio (vs normal control) of the average signal intensities (detection p value < 0.01) were used to construct the dendrogram and the genes and samples were clustered based on Euclidean distance and average linkage method (TIGR MultiExperiment Viewer; [52]). (b) expression of endogenous phospholipases at 24 hour reperfusion (c) expression of endogenous phospholipases at 48 hour reperfusion. Control, sham operated rat brain; MCAo, rats subjected to middle cerebral artery occlusion and reperfused for either 24 hr or 48 hr; PLA, MCAo rat administered with nPLA (0.15 μg/g rats) and allowed reperfusion for 24 hr or 48 hr.