Scorpion Venom Heat-Resistant Peptide is Neuroprotective against Cerebral Ischemia-Reperfusion Injury in Association with the NMDA-MAPK Pathway

Abstract

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Our previous studies have shown that SVHRP is neuroprotective in models of Alzheimer's disease and Parkinson's disease. The present study aimed to explore the potential neuroprotective effects of SVHRP on cerebral ischemia/reperfusion (I/R) injury, using a mouse model of middle cerebral artery occlusion/reperfusion (MCAO/R) and a cellular model of oxygen-glucose deprivation/reoxygenation (OGD/R). Our results showed that SVHRP treatment decreased the neurological deficit scores, edema formation, infarct volume and neuronal loss in the MCAO/R mice, and protected primary neurons against OGD/R insult. SVHRP pretreatment suppressed the alterations in protein levels of N-methyl-D-aspartate receptors (NMDARs) and phosphorylated p38 MAPK as well as some proinflammatory factors in both the animal and cellular models. These results suggest that SVHRP has neuroprotective effects against cerebral I/R injury, which might be associated with inhibition of the NMDA-MAPK-mediated excitotoxicity.

Keywords: Cerebral ischemia/reperfusion injury; NMDARs; Neuroprotection; Scorpion venom heat-resistant peptide; p38 MAPK.

Fig. 1

Effects of SVHRP (20 μg/kg, i.p.) on neurological deficit score, infarct volume, and water content in mice after MCAO/R. A Neurological deficit scores 24 h after MCAO/R. Pretreatment with SVHRP significantly reduced the scores. B Representative coronal brain slices stained with TTC. Red, healthy tissue; white, infarcted tissue. Pretreatment with SVHRP significantly reduced the infarct size. C, D Quantitative analyses of infarct volume (C) and brain water content (D). Pretreatment with SVHRP reduced brain water content after MCAO/R (mean ± SEM; n = 6–16 mice/group; *P < 0.05, ***P < 0.001, one-way ANOVA followed by the LSD post-hoc test).

Fig. 2

Effects of SVHRP on the survival of neurons after I/R injury. A Effects of SVHRP (20 μg/kg, i.p.) on neuronal survival in the ischemic penumbra after MCAO/R. (a) Representative image of Nissl staining in cortex 24 h after MACO/R (scale bars, 500 μm for upper panel, 100 μm for lower panel). (b, c) Density (b) and cell number (c) analyzed by quantitative imaging (Proplus). SVHRP pretreatment attenuated the neuronal loss caused by MCAO/R and SVHRP alone had no effect on these histological changes (mean ± SEM; n = 5 mice/group; *P < 0.05, **P < 0.01, one-way ANOVA followed by Turkey’s test). B Effects of SVHRP (20 μg/mL) on survival of primary cortical neurons after OGD/R. (a, b) Cell viability (a) by MTT assay and LDH release rate (b) from cultured cortical neurons (mean ± SEM; data from 3 separate experiments; ***P < 0.001, one-way ANOVA followed by the LSD post-hoc test).

Fig. 3

Effects of SVHRP on the expressions of NR1, NR2B, NR2A and GABA_A receptors in the ischemic penumbra after MCAO/R and in cultured cortical neurons under OGD/R. A Pretreatment with SVHRP (20 μg/kg, i.p.) significantly decreased the protein levels of NR1 (a) and NR2B (b), and increased the NR2A (c) and GABA_A (d) levels compared with the MCAO/R group. B Primary cortical neurons subjected to ODG/R injury and the effects of SVHRP (20 μg/mL) pretreatment on the protein levels of NR1 (a), NR2B (b), NR2A (c), and GABA_A (d) corresponded with those in the MCAO/R mice. Data are presented as the mean ± SEM; n = 3 mice/group; *P < 0.05, **P < 0.01, one-way ANOVA followed by Turkey’s test.

Fig. 4

Effect of SVHRP on the expression of p-p38 MAPK in MCAO/R and OGD/R models. A Representative immunoblots (a) and densitometric analysis (b) of the immunoblots demonstrated that SVHRP (20 μg/kg, i.p.) decreased the expression of p-p38 MAPK in the ischemic penumbra after MCAO/R. B SVHRP (20 μg/mL) remarkably down-regulated p-p38 MAPK expression in OGD/R-treated cortical neurons, corresponding with the effect in vivo (mean ± SEM; n = 3 mice/group; *P < 0.05, **P < 0.01, one-way ANOVA followed by Turkey’s test).

Fig. 5

Effects of SVHRP (20 μg/kg, i.p.) on the expression of iNOS protein and TNF-α and IL-6 mRNA in the ischemic penumbra after MCAO/R. The MCAO/R group had significantly higher expression of iNOS protein (A) and TNF-α (B), IL-6 mRNA (C) than that of the sham operation groups, and SVHRP pretreatment significantly reduced their levels. There were no significant differences between the NS+sham and SVHRP+sham groups (mean ± SEM; n = 3 mice/group; *P < 0.05, one-way ANOVA followed by Tukey’s test).