Hosphorylation of PI3KAkt and GSK3 is usually a essential step in numerous cellular processes, which include proliferation, development, survival, and apoptosis [58,59]. Previous studies have Cd4 Inhibitors products demonstrated that MPP swiftly and reversibly decreases Akt and GSK3 phosphorylation [31,60], which correlates with increased neuronal death [61,62]. Thus, we evaluated no matter whether PI3KAkt and GSK3 signaling pathways are involved in the antiapoptotic effects of sulfuretin. Constant with prior 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 often 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 drastically inhibits the activity of GSK3 [66]. LY294002 abolished the antiapoptotic effect of sulfuretin by stopping the phosphorylation of Akt and GSK3 (Figure 5A,B). Furthermore, SB415286 attenuated MPP induced apoptosis, mimicking the (R)-(+)-Citronellal supplier protective effects of sulfuretin in SHSY5Y cells (Figure 5C). These benefits demonstrated that PI3KAkt and GSK3 mediates the protective effects of sulfuretin against MPP in SHSY5Y cells. Consistent with our results, it was reported that PI3KAkt is activated by sulfuretin and accountable for th sulfuretininduced protective effect against amyloid [26]. MAPK signaling pathways are involved in a lot of cellular events, such as differentiation, proliferation, and apoptosis, and a minimum of 3 important MAPK subfamilies (ERK, JNK, and p38) have been characterized [67]. Amongst them, ERK increases the survival of dopaminergic neurons [35,68]. The phosphorylation of ERK is reported to be suppressed soon after four h of exposure to MPP in SHSY5Y cells [35]. Our study confirmed that MPP lowered the phosphorylation of ERK, whereas sulfuretin reversed the MPP mediated ERK dephosphorylation (Figure 4B). It has previously been demonstrated that phosphorylated ERK migrates towards the nucleus and regulates different transcription factors, top to adjustments in gene expression and cell proliferation [69]. In distinct, PD98059 abolished the protective effects of sulfuretin on cell viability, suggesting that ERK is vital for sulfuretininduced protectionInt. J. Mol. Sci. 2017, 18,13 ofagainst MPP cytotoxicity (Figure 6A). Interestingly, LY294002 decreased the phosphorylation of Akt and GSK3 without altering ERK phosphorylation. Consistently, PD98059 decreased ERK phosphorylation without having changing phosphorylation of Akt or GSK3 (Figure 6B). These outcomes indicate that each AktGSK3 and ERK contribute for the protective effects of sulfuretin in a mutually independent manner. Similarly, Zhang et al. showed that in principal dopaminergic neurons, valproic acid has protective effects against MPP induced neurotoxicity which include apoptosis, dopamine uptake reduction and tyrosine hydroxylase inactivation [29]. LY294002 and PD98059 reversed valproic acidinduced neuroprotective effects. Interestingly, pretreatment with each LY294002 and PD98059 showed further reverse effects in comparison to LY294002 or PD98059 alone, suggesting additive effect of PI3KAkt and ERK signaling pathways. Even though we didn’t investigate how sulfuretin impacts these signaling pathways, ROS could possibly possess a potential as an up.