Hosphorylation of PI3KAkt and GSK3 is really a important step in a variety of cellular processes, which include proliferation, development, survival, and apoptosis [58,59]. Previous studies have demonstrated that MPP rapidly and reversibly decreases Akt and GSK3 phosphorylation [31,60], which correlates with increased neuronal death [61,62]. For that reason, we evaluated no matter if PI3KAkt and GSK3 signaling pathways are involved within the antiapoptotic effects of sulfuretin. Consistent with previous reports, MPP decreased the phosphorylation of Akt at Ser473 and GSK3 at Ser9; however, sulfuretin reversed the dephosphorylation of Akt and GSK3 in MPP treated SHSY5Y cells (Figure 4A). GSK3 is a downstream target of Akt [63] and an important mediator of MPP induced cell injury [64]. GSK3 activation facilitates mitochondrial dysfunction, whereas its inhibition prevents neuronal loss by suppressing proapoptotic proteins [65]. Phosphorylation of GSK3 at Ser9 is mostly controlled by Akt and this phosphorylation substantially inhibits the activity of GSK3 [66]. LY294002 abolished the antiapoptotic impact of sulfuretin by preventing the phosphorylation of Akt and GSK3 (Figure 5A,B). Additionally, SB415286 attenuated MPP induced apoptosis, mimicking the protective effects of sulfuretin in SHSY5Y cells (Figure 5C). These final results demonstrated that PI3KAkt and GSK3 mediates the protective effects of sulfuretin against MPP in SHSY5Y cells. Constant with our results, it was reported that PI3KAkt is activated by sulfuretin and responsible for th sulfuretininduced protective impact against amyloid [26]. MAPK signaling pathways are involved in several cellular events, like differentiation, proliferation, and apoptosis, and at the very least three significant MAPK subfamilies (ERK, JNK, and p38) have already been characterized [67]. Among them, ERK increases the survival of dopaminergic neurons [35,68]. The phosphorylation of ERK is reported to be A phosphodiesterase 5 Inhibitors Related Products suppressed just after 4 h of exposure to MPP in SHSY5Y cells [35]. Our study confirmed that MPP decreased the phosphorylation of ERK, whereas sulfuretin reversed the MPP mediated ERK dephosphorylation (Figure 4B). It has previously been demonstrated that phosphorylated ERK migrates to the nucleus and regulates various transcription things, top to changes in gene expression and cell proliferation [69]. In certain, PD98059 abolished the protective effects of sulfuretin on cell viability, suggesting that ERK is important for sulfuretininduced protectionInt. J. Mol. Sci. 2017, 18,13 ofagainst MPP Trequinsin Metabolic Enzyme/Protease cytotoxicity (Figure 6A). Interestingly, LY294002 decreased the phosphorylation of Akt and GSK3 without altering ERK phosphorylation. Regularly, PD98059 decreased ERK phosphorylation without altering phosphorylation of Akt or GSK3 (Figure 6B). These benefits indicate that both AktGSK3 and ERK contribute to the protective effects of sulfuretin in a mutually independent manner. Similarly, Zhang et al. showed that in key dopaminergic neurons, valproic acid has protective effects against MPP induced neurotoxicity like apoptosis, dopamine uptake reduction and tyrosine hydroxylase inactivation [29]. LY294002 and PD98059 reversed valproic acidinduced neuroprotective effects. Interestingly, pretreatment with both LY294002 and PD98059 showed additional reverse effects compared to LY294002 or PD98059 alone, suggesting additive impact of PI3KAkt and ERK signaling pathways. Despite the fact that we didn’t investigate how sulfuretin impacts these signaling pathways, ROS could possibly have a possible as an up.