Oxidative stress induces neuronal cell death, which is implicated in many neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis [1–3]. The underlying mechanism, however, is poorly understood. Therefore, in the present study we investigated the possible mechanism by which melittin exerts its protective effects in H2O2-induced SH-SY5Y apoptotic cell death.
Several studies have demonstrated that H2O2-induced apoptotic cell death depends on the concentration and exposure time of H2O2. Gardner et al. demonstrated that moderate concentrations of H2O2 induced DNA cleavage and morphologic changes leading to apoptosis . In the present study, we confirmed that SH-SY5Y cells treated with 0 to 400 μM H2O2 exhibited a dose-dependent loss of cell viability (data not shown). Pretreatment with 0.5 to 1 μg/ml melittin, however, significantly protected cell viability, which was confirmed by the lack of morphologic changes in melittin-pretreated cells. These findings suggest that melittin prevented SH-SY5Y cells from undergoing H2O2-induced apoptotic cell death. Staining the apoptotic nuclei with DAPI revealed that melittin slightly attenuated the induction of apoptotic features, such as cell shrinkage, nuclear condensation and DNA fragmentation, compared with cells treated with H2O2 alone.
Apoptotic cell death in SH-SY5Y cells induced by H2O2 is mediated by mitochondria through intrinsic pathways that activate caspases . The Bcl-2 family contains two groups, an anti-apoptotic group (Bcl-2 and Bcl-x L) and a pro-apoptotic group (Bax, Bid and Bak), and these groups play a crucial role in the mitochondrial-related apoptosis pathway [23, 24]. Anti-apoptotic Bcl-2, which inhibited the release of cytochrome c, is located in the outer mitochondrial membrane . In contrast, pro-apoptotic factor, Bax, resides in the cytosol and translocates to the outer mitochondrial membrane, which might lead to the loss of mitochondrial membrane potential, an increase in mitochondrial membrane permeability and the release of cytochrome c from the intermembrane space into the cytosol, leading to cell death . The Bcl-2 family regulates the apoptotic process through balancing of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) products . In this regard, the Bax/Bcl-2 ratio is suggested to be a useful predictor of apoptotic cell death [24, 27]. In present study, we examined the protein and mRNA expression of Bcl-2 and Bax in H2O2-induced apoptotic cell death in SH-SY5Y cells. Our findings indicate that H2O2 induced changes in the protein and mRNA expression of Bcl-2 family, Bcl-2, and Bax, in SH-SY5Y cells, but pretreatment with melittin enhanced the protein and mRNA expression of Bcl-2 and reduced the protein and mRNA expression of Bax in SH-SY5Y cells. The Bax/Bcl-2 ratio increased after treatment with only H2O2, while pretreatment with melittin inhibited the increase in the Bax/Bcl-2 ratio. These findings suggest that melittin modulates the effect of H2O2 treatment on the protein and mRNA expression of Bcl-2 and Bax.
Caspase 3 acts as an apoptotic executor by activating DNA fragmentation . In apoptotic processes, cytochrome c is released from the mitochondria to the cytosol. The released cytochrome c activates caspase-9 which in turn triggers the activation of caspase-3, which induces cell death . Increased caspase-3 activity is associated with an increase in the Bax/Bcl-2 ratio . Although we did not examine the expression of cytochrome c and caspase-9 in the present study, our findings indicate that H2O2-induced apoptosis was associated with the expression and activation of caspase-3, which led to an increase in the Bax/Bcl-2 ratio. Pretreatment with melittin, however, inhibited the expression and activation of caspase-3, suggesting that melittin has potential anti-apoptotic effects by modulating the H2O2-induced protein and mRNA expression of Bcl-2 and Bax by downregulating caspase-3 protein expression and activation.
Melittin is a residue of the main toxic compound in honeybee venom, and is a small linear peptide composed of 26 amino acids [13, 14]. Although melittin is a toxic peptide, several studies have demonstrated various properties of melittin, including anti-bacterial, anti-arthritic, and anti-inflammatory effects [14–16]. Pratt et al. reported that melittin (2 μM) did not disrupt cell membranes of leukocytes . Also, a recent study showed that a lower dose of melittin (0.5 and 1 μg/ml) protected hepatocytes against TGF-β1 .