Protective Effects Of Polysaccharide From Spirulina Platensis Against MPTP-induced Dopaminergic Neurodegeneration In Mice And MES 23.5 Cells

Parkinson's disease (PD) is a disorder characterized by the degeneration ofdopaminergic neurons in substantia nigra (SN). Recent advances suggest that dysfunctionof the mitochondrial respiration and generation of free radicals by oxidative catabolism ofdopamine (DA) is important mediator of neuronal death in PD. The neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) recreates a PD-like model in bothrodents and primates. MPTP neurotoxicity depends on the monoamine oxidase B(MAO-B)-catalyzed production of MPP~+ which is taken up selectively by dopaminergicneurons and concentrated into the mitochondria. MPP~+ inhibits the activity of complexⅠof the electron transport chain leading to the production of reactive oxygen species, theloss of the mitochondrial membrane potential (Δψm) and ATP production, and theneuronal death. MPP~+ also induces DA efflux, increasing DA autoxidation and oxidativedamage. Therefore, enhancing the activity of free radical scavengers system anddecreasing the DA turnover rate become the new treatment strategies of PD.Polysaccharides is an important biomacromolecule besides protein and nucleic acid,which plays important role in control cell division and differentiation, modulate cellgrowth, delay aging, maintain the metabolism, and so on. Polysaccharide from SpirulinaPlatensis (PSP) is a kind acor heteropolysaccharide extracted form Spirulina Platensis. Inprevious studies, PSP showed a variety of biological properties such as anti-aging,antiradiation, anti-tumor, antioxidation, and enhancement immune functions. While theprotective effect of PSP on dopaminergic neurons is still unknown. In the present study,immunohistochemistry assay, reverse transcription polymerase chain reaction (RT-PCR), HPLC-ECD were used to detect the protective effect and the possible mechanism of PSPon dopaminergic neurons of MPTP-induced PD mice model; detected the influence ofPSP on the activities of MAO-B, SOD, GSH-px in mice. MTT assay, FCM, and LSCMwere used to detect the effect of PSP on the viability of MPP~+-damaged MES 23.5 cells,theΔψm changes, the ROS level, and the concentration of free calcium in cells. Theresults were as follows:1. Compared with control group, the TH and DAT immunoreactive stain inMPTP-treated mice were significantly decreased (P＜0.01), while in PSP 800 mg/kgpretreatment group, the immunoreactive staining of TH and DAT in the substantianigra (SN) were increased compared with MPTP group (P＜0.01);2. Compared with control group, the expression of TH mRNA and DAT mRNA ofMPTP-treated mice were significantly decreased in the SN (P＜0.01), while in PSP800mg/kg pretreatment group, the expression of TH mRNA and DAT mRNA wereincreased compared with MPTP group (P＜0.01);3. Compared with control group, the content of DA, DOPAC and HVA in the striatum(Str) of MPTP-treated mice was decreased (P＜0.01), the turnover rate of DA(DOPAC+HVA/DA) was increased (P＜0.01). While in PSP 800mg/kg pretreatmentgroup, the content of DA, DOPAC and HVA in the Str was increased, the turnoverrate of DA was decreased compared with MPTP group (P＜0.01). The resultssuggested that PSP plays a protective effect on MPTP-damaged dopaminergicneurons of C57BL/6J mice;4. The activity of MAO-B in deprenyl pretreated mice was decreased (P＜0.01), whilethere was no significant difference among MPTP-treated group, PSP pretreatmentgroup, PSP treated alone group, and control group; 5. The activities of SOD and GSH-px were decreased significantly in MPTP-treatedgroup compared with the control (P＜0.01); the PSP and deprenyl pretreatment greatlyenhanced the activities of SOD, GSH-px, compared with MPTP-treated group. Suchresults suggested that the antioxidation properties of PSP is the underlyingmechanism of its neuroprotective effect;6. MPP~+ decreased the viability of MES 23.5 cell dose-dependently, in 20μmol/L orhigher concentration, the viability was decreased significantly (P＜0.01). The MES23.5 cell was incubated with 20μmol/L MPP~+ plus different concentration of PSP,the viability increased significantly in 40mg/L or higher concentration of PSPcompared with MPP~+ group;7. 28.8%±5.4% MES 23.5 cells was negative staining by R 123 24 h after 20μmol/LMPP~+ incubation, suggested 28.8% cells were with disfunctionΔψm, the rate washigher than that in control (21.3%±3.3%). In PSP pretreatment group, there was20.1%±3.2% R 123 negative staining cells, the rate was lower than that in 20μmol/L MPP~+ group (P＜0.01); the average fluorescence intensity of R 123 inMPP~+-treated cells was 30.02±3.5, much lower than that in control(46.67±5.3,(P＜0.01), the average fluorescence intensity in PSP 40 mg/L treated cells was43.00±4.7, much higher than that in MPP~+ group (P＜0.01);8. To detect the ROS level in MES 23.5 cells, 46.3%±8.6% MES 23.5 cells waspositive staining by DCF 24h after incubation with 20μmol/L MPP~+, much morethan that in control group (30.08%±5.3%). In PSP 40 mg/L pretreatment group,there was 35.68%±5.7% DCF positive staining cells, the rate was lower than that in20μmol/L MPP~+ group (P＜0.05); the average fluorescence intensity of DCF inMPP~+-treated cells was 64.07±10.6, much higher than that in control(31.8±5.7,(P＜0.01), the average fluorescence intensity in PSP 40mg/L treated cells was 38.20±4.2, much lower than that in MPP~+ group (P＜0.01);9. To detect the concentration of free calcium in MES 23.5 cells, the averagefluorescence intensity of Fluo-3 in 20μmol/L MPP~+ incubation cells was muchhigher than that in control group (160.28±48.99 vs 57.32±13.52); In PSP 40mg/Lpretreatment group, the average fluorescence intensity was 83.03±8.22, much lowerthan that in 20μmol/L MPP~+ group (P＜0.01).The results suggest that PSP showed protective effect against MPTP-inducedneurodegeneration in C57BL/6J mice, increased the content of DA and its metabolites inthe Str, decreased the turnover rate of DA. Such protective effect didn't rely on theinhibition of MAO-B, but depended on the antioxidation effect of PSP. Meanwhile, PSPmitigated the damage of MES 23.5 cell from MPP~+. The results showed in this paper arehelpful for understanding the Marine medicine PSP and for developing PSP to be one ofthe new therapeutic strategies for PD.