Ethanol exposure during prenatal development causes a wide range of structural and functional brain abnormalities [30, 31]. It has already been established that ethanol depletes many cell populations during development, but not all populations of neurons in the brain are susceptible; furthermore, ethanol increases neuronal apoptosis, which causes malformations, including the loss of brain mass [1, 32, 33]. Ethanol increases ROS generation, which can trigger apoptotic cell death pathways through the activation of caspases [34–36]. The present study provides strong evidence for a direct neuroprotective role of Met and TQ. The current investigation indicated that cotreatment of ethanol with Met, TQ or Met plus TQ protects cortical neurons from the apoptosis triggered by ethanol during early development.
The evidence regarding neuroprotection with the antidiabetic drug Met is supported by studies that state its beneficial effects in various models of toxicity. It was reported that Met can protect against neurotoxicity, in addition to its antidiabetic effect, and it has a number of important, diverse functions, including in polycystic ovarian syndrome, induction of osteoblasts via AMP-activated protein kinase (AMPK) signaling pathway along with subsequent enhancement of nitric oxide synthase (eNOS) and bone morphogenetic-2 protein (BMP-2) production [37, 38]. Met modulates AMPK and mitogen-activated protein kinase (MAPK) pathways to induce bone formation, which mimics the intracellular metabolic changes associated with nutritional deprivation [39, 40]. An additional target of Met is the respiratory chain complex I of the mitochondria, which activates AMPK via changes in mitochondrial reactive nitrogen species [41, 42]. However, we provide here a new and potential mechanism that supports a protective role of Met on apoptosis that is likely due to it maintaining mitochondria integrity and reducing [Ca2+]c overload as well as subsequent cell death .
Furthermore, we studied TQ, which is a natural compound with different beneficial pharmacologic effects against various cytotoxic insults. These beneficial effects have been observed in vitro and in vivo with degeneration caused by L-dopa in SH-SY5Y human neuroblastoma cells, serum/glucose deprivation induced cell death (SGD) and chronic toluene exposure [21, 22, 43]. In general, previously published reports showed that TQ mainly functions through its antioxidant mechanism, and it has been used as a protective agent in multiple toxicity models, such as doxorubicin-induced cardiotoxicity, cisplatin-induced nephrotoxicity and ischemic neuronal cell death [21, 23, 44].
We found that administration of Met and TQ in selected doses has the capability to maintain normal cell viability against ethanol. TQ, Met or a combination of both reduced the cell death induced by ethanol (Figure 1A, B). Calcium homeostasis is an important for normal cellular physiological function, and even a minute variation in normal levels can provoke abnormal cellular function and cell death . This [Ca2+]c overload further disrupts mitochondrial membrane potential (ΔψM), which leads to neuronal apoptosis. In this study, we observed a two-fold increase in [Ca2+]c levels upon ethanol treatment compared to control cells. This cellular accumulation of calcium possibly induced cell death. Previously, a similar increase in intracellular [Ca2+]c was observed after a short (20 min) exposure to ethanol in prenatal primary neuronal cells , fetal hypothalamic cells and cervical ganglion neurons [45, 46]. TQ and Met reduced elevated [Ca2+]c levels (Figure 2). TQ and Met modulate and stabilize ΔψM. Our findings that showed ethanol-induced alterations in mitochondrial function were also in agreement with a recent study that demonstrated that vitamin E and BDNF have neuroprotective and modulating roles during ethanol toxicity in cerebellar granule cells. The mechanism of neuroprotection was similar to Met and TQ involved the stabilization of mitochondrial membrane potential and inhibition of apoptotic cascade .
The Bcl-2 protein family plays an important role in apoptotic signal transduction by regulating mitochondrial function . It was reported that mitochondrial cytochrome-c is released into the cytosol through the permeability transition pore, which is regulated by Bcl-2 family proteins . The pro-apoptotic protein Bax destabilizes ΔψM and facilitates cytochrome-c release , while the anti-apoptotic protein Bcl-2 functions to restore membrane potential and block cytochrome-c release. In our findings, we found that TQ and Met increased Bcl-2 levels in primary cortical neurons exposed to ethanol. TUNEL and western blot analysis demonstrated that TQ and Met cotreatment protects neurons by lowering the expression of caspase-3, cytochrome-c, cleaved caspase-9 while also increasing the expression of Bcl-2 compared to cells treated with ethanol alone. This finding implied that TQ and Met potentially prevents apoptosis by regulating the mitochondrial path way.
PARP-1 functions in apoptosis by acting as a survival factor and a death promoter, depending on the duration and severity of DNA damage . We observed cleavage of PARP-1 to its respective 89 kDa fragment after a 12 h exposure to ethanol. In Met and TQ treatment groups, fragmentation of PARP-1 was significantly reduced. In support of our results, other investigators noted caspase-dependent cleavage of PARP-1 that was induced by an ethanol concentration (4.0 mg/ml), similar to the one used in this study, for 0, 4, 12 and 24 h time periods . The potential benefit of Met and TQ on primary neuronal cell viability was evaluated by TUNEL assay. Met and TQ decreased DNA damage and reduced the number of TUNEL-positive cells produced by ethanol treatment. A study using an analogous experimental approach demonstrated similar DNA damage induced by ethanol in cultured cortical neurons .
The present findings supported a conclusion that ethanol disrupts ΔψM, sustains Ca2+ overload and triggers an apoptotic cascade. Met and TQ inhibition of ethanol-induced cell death in primary rat cortical neurons might occur via an antioxidant mechanism that maintains mitochondrial integrity. Since Met and TQ are safe and nontoxic, more studies should be conducted to explain their synergistic actions with other drugs.