Neuroprotective Effects Of Tetrahydroxystilbene Glucoside In The MPTP Mouse Model Of Parkinson’s Disease Through Activation Of PI3k/AKt Pathway

Parkinson’s disease (PD) is a common neurodegenerative disorder that ischaracterized by the progressive loss of dopaminergic neurons in the substantianigra and dopamine depletion in the striatum. Even though the etiology of PDremains unclear, several lines of evidence strongly suggest that oxidative stressand mitochondrial dysfunction play an important role in the neurodegenerativeprocess of this disease. The most classic therapy for PD is levodopaadministration, but the efficacy of levodopa treatment declines as the diseaseprogresses. The neuroprotective strategies to rescue nigral dopamine neuronsfrom progressive death are currently being explored, and among them, theChinese herbs and herbal extracts have shown potential clinical benefit inattenuating the progression of PD in human beings.Polygonum multiforum Thunb (PM) is a traditional Chinese herb that hasbeen used widely in the Orient since ancient times as a tonic and anti-agingagent. In clinical, we found the herbal prescription which is “gentleman medicine” of PM have certain curative effect for PD treatment. It has beenreported that PM extract (PME-Ⅰ) had neuroprotective effects and behavioralimprovement in the mouse model of neurotoxin-induced DAergic neuronaldamage.2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) is one of themain active ingredients of PME-Ⅰ, which has a range of pharmacologicalproperties, including antioxidative, anti-infammatory and inhibiting oxidativestress effects.1-methyl-4-phenylpyridinium (MPP+), the active metabolite of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), can selectively damage DAergicneurons and resulted in a depletion of dopamine in the nigrostriataldopaminergic pathway, which caused a parkinsonian syndrome in monkey ormice. Our in vitro experiments suggested that TSG affords a signifcantneuroprotective effect against MPP+-induced damage and apoptosis in PC12cells though activation of the PI3K/Akt pathway. In order to further research theeffects of TSG in vivo, we have investigated the effects of TSG on behavioraland neurohistological alterations in mice caused by treatment of MPTP.Moreover, we sought to determine whether neuroprotection by TSG againstMPTP is mediated by the activation of the PI3K/Akt pathway.【Objective】1. Establish the subcute MPTP mouse model of PD.2. Investigate the effects of TSG on behavioral and histochemical alterations inMPTP-treated mice model of PD.3. Determine whether neuroprotection by TSG against MPTP is mediated by theactivation of the PI3K/Akt pathway.【Methods】1. The effects of TSG on MPTP-induced parkinsonism were studied in the following experimental groups:(A) NS (saline, i.p.)+NS (i.g.);(B) MPTP (i.p.)+NS (i.g.);(C) MPTP (i.p.)+TSG20mg/kg/day (i.g.);(D) MPTP (i.p.)+TSG40mg/kg/day (i.g.); and (E) NS (i.p.)+MPTP (i.p.)+TSG40mg/kg/day (i.g.)+LY2940023.75mg/kg/day (i.g.). Animals in groups A-E received saline orMPTP (30mg/kg i.p.) daily for7days, vehicle (saline) or TSG treatment (i.g.)was started1h after the last MPTP administration and given daily for14days.The behavioral changes of mice were detected by pole test and grid test.2. Mice were killed by cervical dislocation or perfusion after the last vehicle orTSG administration and behavior observation. The TH and DAT-positiveneurons in the substantia nigra were observed by immunofluorescencehistochemistry.3. The striatal TH, DAT, Akt, phospho-Akt (Ser473), GSK3β, phospho-GSK3β(Ser9), Bcl-2/BAD, caspase-9and caspase-3were detected by Western blotting.【Results】1. In the pole test, the animals which had received repeated treatment withMPTP showed significantly prolonged Tturn(P<0.01) and TLA(P<0.01) ascompared to the control animals which had received saline alone during thesame period, indicating an induction of bradykinesia. Pretreatment of TSG (20or40mg/kg, i.g.) significantly reversed the MPTP-induced prolongation of Tturn(P<0.05) and TLA(P<0.05; P<0.01).2. Immunohistochemistry staining demonstrated that nearly all THimmunoreactive neurons were double labeled with DAT, indicating they containthe machinery to be functional DAergic neurons in the mice SN. There weresignificant reductions in the number of TH and DAT-positive neurons in SNpcfor the MPTP group compared with the control group (34.2±2.77,32.2±1.89,P<0.01). In mice receiving daily oral administration of40mg/kg TSG the number of TH and DAT-positive neurons was significantly higher than those inMPTP-vehicle treated mice (67.3±2.79,67.3±2.11, P<0.01), and it was evenclose to those of the control group. A similar but weaker effect was also seen inthe20mg/kg TSG treatment group (45.8±2.06,39.4±0.98, P<0.05).3. Pretreatment of TSG protected dopaminergic neurons by preventingMPTP-induced descreased striatal TH and DAT protein levels. Furthermore, italso increasing striatal Akt and GSK3β phosphorylation, up-regulation of theBcl-2/BAD ratio and inhibiting the activation of caspase-9and caspase-3.【Conclusion】1. TSG improve the behavioral, reduce the DAergic neurons damage in theMPTP mouse model of PD in a dose manner.2. Neuroprotection by TSG against MPTP is mediated by the activation of thePI3K/Akt pathway.