Dl-3-n-Butylphthalide Protects Dopamine Neurons In Rotenone Models For Parkinson's Disease

Objective: To explore whether dl-3-n-Butylphthalide (NBP) has protective effects on rotenone-induced parkinsonian models in vitro and in vivo, and the possible underlying mechanisms.Methods: Rotenone-treated human doparminergic neuroblastoma SH-SY5Ycells were employed for the in vitro experiments. The cells were pretreated with 0.1μM, 1μM, 10μM and 100μM NBP respectively for 24 hours prior to rotenone administration. The cell morphology was observed; cell viability was detected by MTT; Annexin V-FITC/ propidium iodide staining was used to analyze the apoptosis; mitochondrial membrane potential was detected by JC-1 staining; reactive oxygen species contents was tested by DCFH-DA staining. The in vivo experiment was conducted in Sprague-Dawley rats. Rotenone was stereotaxically infused to the substantia nigra (SNc) and ventral tegmental area (VTA) of the rats to produce PD animal models. 80 mg/kg/day NBP was intragastrically treated daily for 14 days. The numbers of rotations induced by apomorphine (APO) were counted within 30 minutes. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) with the mesencephalon were measured by spectrophotometers. The tyrosine hydroxylase (TH) and Neuronal Nuclei (NeuN) positive cells were detected by double- immunohistostaining. For the mechanism exploring, 100μM NBP or equal volume of DMSO was administrated to four human neuroblastoma cell lines [SH-SY5Y, IMR-32, SK-N-AS, and BE(2)-M17] for 24 hours, and eight rats of each group were intraperitoneally injected with NBP (80 mg/kg, NBP: DMSO: PBS = 1:2:17) or vehicle (DMSO : H2O = 3:17) twice, 18 h and 6 h before perfusion and brain harvest. Finally, vesicular monoamine transporter 2 (VMAT2) andα-synuclein (SNCA) mRNA and protein levels from these four cell lines and SNc of the animals were detected by immunohistostaining and qRT-PCR, respectively.Results: In the experimental cellular model, cell viability loss and apoptosis was induced by rotenone SH-SY5Y cells. NBP pretreatment attenuated the cell viability loss and apoptosis induced by rotenone. Compared to rotenone group, the mitochondrial membrane potential was markedly elevated (P<0.05), and the levels of reactive oxygen species were significantly declined (P<0.05) in the NBP-treated groups. Importantly, the protective effect of NBP was more significant at higher concentration. In the rodent model, 2-week treatment with NBP was able to ameliorate apomorphine-evoked rotations by 48% (compared to the ipsilateral midbrain of Rot+vehicle-group,P<0.05) and rescue dopaminergic (DA) neurons by 30% (compared to the ipsilateral midbrain of Rot+vehicle-group,P<0.05) and striatal DA terminal by 49% (compared to the ipsilateral midbrain of Rot+vehicle-group,P<0.05). Furthermore, NBP up-regulated VMAT2 expression both in vitro and in vivo. In all cell lines and rats tissues, mRNA and protein levels of SNCA showed no difference between NBP and vehicle administrated.Conclusion: NBP could ameliorate the injury induced by rotenone in PD models. Mitochondrial function reservation, reducing oxidative stress and the subsequent anti-apoptotic effect, VMAT2 up-regulation may be the underlying mechanisms for the neuroprotective effects of NBP. Moreover, NBP could up-regulate the expression of the DA neurons-protective VMAT2 without inducing SNCA over-expression. These findings suggest that NBP, a novel agent for clinical treatment of stroke, could be a potential neuroprotective alternation for PD.