Role of Rutin in 1-Mtthyl-4-Phenylpyridinium toxicity: Therapeutic implications for Parkinson's disease

Abstract

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Although the etiology of PD is not completely known, it is believed to involve an association of various genetic, cellular, and environmental factors that individually or simultaneously advance neuronal degeneration. Neurotoxins such as 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) have been widely used to investigate distinct underlying mechanisms involved in the pathogenesis of PD. Presently, treatment options for PD are limited, as the available drugs are mainly focused on alleviating symptoms with limited ability to prevent disease progression. Accordingly, there is an increase in the use of natural compounds/products as potential neuroprotective agents. These neuroprotective treatments are believed to intervene in some stages in the pathogenesis of PD to suppress possible mechanisms of dopaminergic neuronal death such as apoptosis, mitochondrial dysfunction, oxidative stress, disturbances of calcium homeostasis, inflammation and autophagy. Thus, novel protective strategies for PD may be designed by targeting these mechanisms or intracellular signaling cascades that participate in PD pathogenesis. Plant-derived bioactive compounds used in traditional medicine have beneficial effects on some disorders including PD. For example, the bioflavonoid rutin, derives its common name from Ruta graveolens, a plant that contains high amounts of rutin. It is present in over 130 registered commercially available medicinal preparations and pharmacological studies have reported the beneficial effects of rutin in many disease conditions. Although rutin has been found to attenuate 6 OHDA toxicity in PC12 cells, its activity in MPP+-treated SH-SY5Y cells and fibroblasts have not been investigated. Consequently, for the first time, the protective activity of rutin in MPP+-treated SH-SY5Y cells and primary dermal fibroblasts was investigated, thus revealing possible molecular pathways and mechanisms of action. Findings from the cell viability studies show that rutin significantly protected SH-SY5Y cells and primary dermal fibroblasts from MPP+ toxicity. Additional findings further revealed that rutin pretreatment significantly attenuated MPP+ triggered increase in the production of nitric oxide (NO) and reactive oxygen species (ROS) in SH-SY5Y cells. The attenuation of increased ROS and NO production in SH-SY5Y cells is a crucial mechanism of action for its protection against MPP+ induced DNA damage and inflammation. This was demonstrated by a significant reduction in the expression levels of DNA damage (γH2AX) and inflammation (COX-2) markers following rutin pretreatment in SH-SY5Y cells. Also, rutin significantly suppressed MPP+ induced disruption of antioxidant enzymes and prevented MPP+ induced damage in nuclear morphology, clearly evidenced by fluorescence images from Hoechst staining showing shrinkage and fragmentation of SH-SY5Y cells. Meanwhile, the inhibition of p-Akt and p-NF-κB, as well as the activation of p-AMPK in MPP+ treated SH-SY5Y cells resulted in a cascade of apoptotic, autophagic and endoplasmic reticulum (ER) stress events leading to cell death. The ability of rutin to effectively regulate cell signaling pathways could be responsible for the protection of SH-SY5Y cells from the deleterious effects of apoptosis, autophagy and ER stress. This was demonstrated by a significant increase in the expression of full-length caspase 3 and GRP78/BiP, as well as a significant reduction in the expression levels of cleaved PARP, cytochrome C, LC3-II, p62 and CHOP proteins in pretreated SH-SY5Y cells. In confirmation of the western blot findings on autophagy, transmission electron images revealed abnormal presence/accumulation of numerous autophagosomes in our MPP+ treated SH-SY5Y cells while there was significantly reduced autophagic vacuoles in SH-SY5Y cells pretreated with rutin, perhaps due to the capacity of rutin to enhance efficient and speedy clearance of these vacuoles. Furthermore, increased levels of Ca2+ and significantly reduced mitochondrial membrane potential in SH-SY5Y cells, as well as significantly reduced maximal respiration and spare respiratory capacity in SH-SY5Y cells and fibroblasts, clearly highlights major characteristics of mitochondrial dysfunction in cells treated with MPP+. However, these effects were significantly attenuated following rutin pretreatment in SH-SY5Y cells and fibroblasts. Additionally, in SH-SY5Y cells, our findings show that rutin significantly improved basal and compensatory glycolysis as a compensatory response to an impaired oxidative phosphorylation system triggered by MPP+ which resulted in an insufficient ATP production. Taken together, proper regulation of the ROS-NO and cell signaling pathways, maintenance of Ca2+ homeostasis, mitochondrial protection and efficient autophagy clearance may account for the neuroprotective effects of rutin observed in our dopaminergic SH-SY5Y cells and fibroblasts. These findings further suggest that rutin may be a promising neuroprotective agent for the treatment of PD. Future studies will involve investigating its activity in animal models of PD.