The present set of in vitro
experiments characterized the effects of Pycnogenol® on a variety of oxidative stress markers. PYC, a patented combination of bioflavonoids, scavenges oxidants and thus promotes cellular health. PYC is a mixture of polyphenols, that decrease toxic byproducts of lipid peroxidation such as H2
]. Previous studies have reported increased SH-SY5Y cell survival with PYC [21
]. The present study demonstrates that PYC can protect SH-SY5Y cells from cytotoxicity induced by acrolein in part by (1) reducing ROS production; (2) remediating suppressed NADPH-oxidase, and (3) enhancing the GSH system [25
]. It is been suggested that PYC may not only suppress the generation of ROS, but also attenuate apoptotic neuronal death in Aβ-induced animal models of AD, possibly by decreasing free radical generation [11
]. Data from this present study shows that treatment with acrolein results in a significant increase of ROS level in a dose-dependent manner. This is consistent with previous descriptions of acrolein-induced oxidative stress [33
GSH protects cultured neurons against oxidative damage resulting from acrolein, and 4-HNE [33
]. GSH can also protect the brain from damage by peroxynitrite, hydroxyl free radicals, or reactive alkenals [42
]. These findings suggest that these changes, are a common feature of various types of oxidative and nitrosative stress, which may be associated with the protein damages, modification of redox regulation and cellular function [43
]. There is evidence that oxidative stress, including free radicals, plays a key role in AD and PD [8
]. Brain membrane lipids are rich in polyunsaturated fatty acids, which are especially sensitive to free radical-induced lipid peroxidation [45
Several studies have reported that increased endogenous antioxidant levels, resulting from dietary or pharmacological intake of antioxidants precursors or substrates, can protect GSH from oxidative depletion and consequently protect the brain against oxidative stress [47
]. PYC has been used as a nutritional supplement to be taken orally, and studies have indicated that this phytochemical is indeed being absorbed and metabolized by humans [50
]. Other studies indicate that PYC pretreatment is associated with the protection of cells undergoing oxidative stress associated death, with potential applications in neurodegenerative diseases [24
Acrolein is the strongest electrophilic aldehyde in nature [29
] that occurs as a ubiquitous pollutant in the environment. It is also formed in vivo
from cellular lipid peroxidation [52
], and reacts with various biomolecules including proteins, DNA, and phospholipids, with the potential to disrupt their function especially in neurodegeneration [29
]. The mechanisms of how acrolein inflicts tissue damage are not fully established, but data suggests that it can induce oxidative stress [54
] (). It has been reported that the acrolein-induced damaging molecules strongly react with GSH, reducing levels and subsequently induces the production of ROS [40
]. Depletion in the GSH defense favors an increase in apoptosis and a decrease in cell survival [36
] (). In the presence of acrolein, NADPH-oxidase can also be an important source of ROS over-production and protein modification, leading to neuronal oxidative stress. It is known that the thiol (-SH)-reactive property of acrolein are responsible for the activation of NADPH-oxidase complex and the inducible nitric oxide synthase (iNOS) [60
]. In the present set of experiments, acrolein significantly increased ROS production, O2−.
levels and activation of O2−.
producing enzyme NADPH-oxidase (Fig. & ). Such events have been linked to apoptotic cell death via caspase-3 activation, DNA fragmentation, and poly (ADP-ribose) polymerase (PARP) cleavage [11
]. PYC suppressed both ROS and NADPH oxidase activation (assembling of cytosolic subunits (p67Phox
) to the membrane subunits (gp91Phox
)), while also attenuating GSH depletion, protein damages, and acrolein-induced cell death.
The results shown in this in vitro study demonstrate that PYC probably acts as an antioxidant at the maximal dose 50 μg/ml. We demonstrated that PYC protected SH-SY5Y cells from acrolein-induced oxidative stress by decreasing the percentage of ROS production and as well as reducing NADPH-oxidase activation. In addition, we also found that PYC attenuated the reduction in GSH levels, by the decline in oxidative/nitrosative burden, which was a normal consequence of acrolein exposure. But the elevation in GSH was not due to its synthesis; since PYC treatment was not effective even at 200 μg/ml. Lower concentrations, were not enough to remove acrolein induced oxidative stress, while high concentrations of PYC, being a xenobiotic for the system, might itself be creating a burden, that would favor oxidative stress in the presence of other oxidants. This may explain why, at higher concentrations (100μg/ml) PYC did not provide sufficient antioxidant defenses. Data showing PYC protects SH-SY5Y cells from acrolein cytotoxicity, suggests that the mechanism of acrolein neurotoxicity involves oxidative stress, and that the neuroprotective effects of PYC are associated with its antioxidant properties. We also observed that acrolein may have destructive effects through mechanisms other than oxidative stress, since pretreatment with PYC never resulted in values equivalent to that of controls. PYC could be a key component for the development of therapeutics for neurodegenerations including AD, but case in its effective concentrations and timings for treatment must be observed.