Simvastatin induces neuroprotection in 6-OHDA-lesioned PC12 via the PI3K/AKT/caspase 3 pathway and anti-inflammatory responses

Abstract

Background: In addition to their original applications for lowering cholesterol, statins display multiple neuroprotective effects. Inflammatory reactions and the PI3K/AKT/caspase 3 pathway are strongly implicated in dopaminergic neuronal death in Parkinson's disease (PD). This study aims to investigate how simvastatin affects 6-hydroxydopamine-lesioned PC12 via regulating PI3K/AKT/caspase 3 and modulating inflammatory mediators.

Methods: 6-hydroxydopamine-treated PC12 cells were used to investigate the neuroprotection of simvastatin, its association with the PI3K/AKT/caspase 3 pathway, and antiinflammatory responses. Dopamine transporters (DAT) and tyrosine hydroxylase (TH) were examined in 6-hydroxydopamine-treated PC12 after simvastatin treatment.

Results: Simvastatin-mediated neuroprotection was associated with a robust reduction in the upregulation induced by 6-OHDA of inflammatory mediators including IL-6, COX2, and TNF-α. The downregulated DAT and TH levels in 6-OHDA-lesioned PC12 were restored after simvastatin treatment. Simvastatin reversed 6-OHDA-induced downregulation of PI3K/Akt phosphorylation and attenuated 6-OHDA-induced upregulation of caspase 3 in PC12. Furthermore, the PI3K inhibitor LY294002 pronouncedly abolished the simvastatin-mediated attenuation in caspase 3.

Conclusions: Our results demonstrate that simvastatin provides robust neuroprotection against dopaminergic neurodegeneration, partially via antiinflammatory mechanisms and the PI3K/Akt/caspase 3 pathway. These findings contribute to a better understanding of the critical roles of simvastatin in treating PD and might elucidate the molecular mechanisms of simvastatin effects in PD.

Figure 1

(A–C) Simvastatin reduced the 6‐OHDA‐mediated elevation in the expressions of IL‐6, TNF‐α, and COX2. 6‐OHDA pronouncedly increased the expression of IL‐6, TNF‐α, and COX2 compared with the controls (**P < 0.001, A–C). Simvastatin significantly abolished the elevation (††P < 0.01, A–C). The simvastatin + 6‐OHDA treatment produced significant increases in the expressions of IL‐6, TNF‐α, and COX2 (##P < 0.01, A–C) compared with the controls. D–E. Simvastatin attenuated the 6‐OHDA‐mediated decrease in TH and dopamine transporter (DAT) mRNA levels. Compared with the controls, the 6‐OHDA incubation pronouncedly decreased the TH and DAT mRNA (**P < 0.01, D–E); this decrease was significantly prevented following simvastatin treatment (†P < 0.05, D–E). All of the results are expressed as the mean ± SEM.

Figure 2

Effects of 6‐OHDA and simvastatin on TH and dopamine transporter (DAT) immunocytochemistry staining in PC12 cells. The arrow indicates a positive cell. (A–D) show the TH staining in the PC12 cells of the control, simvastatin, 6‐OHDA, and 6‐OHDA + simvastatin groups, respectively. (E–H) show the DAT staining in the control, simvastatin, 6‐OHDA, and 6‐OHDA + simvastatin groups, respectively. Bar = 100 μm. (I) represents the average number of TH‐positive PC12 cells of the control, simvastatin, 6‐OHDA, and 6‐OHDA + simvastatin groups, respectively. (J) represents the average number of DAT‐positive PC12 cells of each group. The pattern of alteration for DAT and TH immunocytochemistry staining is consistent with the real‐time PCR results.

Figure 3

Effect of 6‐OHDA and simvastatin on the levels of PI3K, Akt, and caspase 3. Simvastatin significantly increased the protein levels of phosphorylated PI3K and Akt compared with the controls (^oo p<0.01, A, B). 6‐OHDA incubation significantly decreased the levels of phosphorylated PI3K and Akt compared with the controls (**P < 0.01, A, B), but this reduction was reversed by simvastatin treatment (††P < 0.01, A, B). 6‐OHDA pronouncedly increased the protein levels of caspase 3 (**P < 0.01, C). This increase was partially inhibited by simvastatin treatment (††P < 0.01, C). However, coincubation with simvastatin and LY294002 partially attenuated the effect of simvastatin alone (&&P < 0.01, C). All of the results are expressed as the mean ± SEM.

Figure 4

Simvastatin protected PC12 cells against 6‐OHDA neurotoxicity. (A) The CCK‐8 value in the 6‐OHDA‐treated group was significantly reduced compared with the controls (**P < 0.01), but simvastatin attenuated this reduction (††P < 0.01). (B–F) Simvastatin protected PC12 cells against 6‐OHDA‐induced apoptosis. (B) represents the apoptosis rate for different groups. (C–F) represent controls, simvastatin, 6‐OHDA‐treated, and 6‐OHDA + sim‐treated group, respectively. 6‐OHDA induced significant apoptosis (**P < 0.01, controls vs. 6‐OHDA; B–F), but simvastatin incubation attenuated this apoptotic cell death (6‐OHDA vs. 6‐OHDA + sim, ††P < 0.01; 6‐OHDA + sim vs. controls, ##P < 0.01, B–F). All of the results are expressed as the mean ± SEM.