Protective Effects Of2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside In The MPTP Mice Model Of Parkinson’s Disease: Involvement Of ROS Mediated Mitochondrial And JNK, P38Pathways

Parkinson’s disease (PD) is a kind of progressive movement disorder in older peoplethat characterized by akinesia, rigidity, tremor, bradykinesia and even depression. Theselective lose of dopaminergic neurons of substantia nigra pars compacta (SNpc) have beenknown as the pathological factor of PD. Current treatments for PD can’t slow or halt theprogression of the disease. The existing medications are symptomatic treatment, and theefficacy of medicine is attenuated. The etiology of PD is still not clear, but convincingevidence demonstrates mitochondrial dysfunction and oxidative stress (OS) constitute themost prominent features found in PD. In addition, increasing evidence points to that themitogen-activated protein kinase (MAPK) pathway signaling is involved in dopaminergicneurons degeneration. Blockade of c-Jun N-terminal kinases (JNK) and p38in cultures hasbeen shown to protect dopaminergic neurons from neurotoxicity.(1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine, MPTP) has been widely used as a neurotoxin for inducing mouse model of Parkinson’s disease, and exerts its neurotoxicitythrough its active metabolite,1-methyl-4-phenyl pyridium (MPP+). Inside dopaminergicneurons, MPP+can impair mitochondrial respiration and inhibiting complex I of the electrontransport chain. This pathological disorders cause severe loss of neuron in substantia nigraand striatal dopamine depletion by overproduced ROS.He shou wu is a famous traditional Chinese medicinal herb and has been long used asa tonic and antiaging agent in Asia. A monomer of stilbene,2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), is one of the main activeingredients of PM. TSG has been reported to possess many biological activities, such asantioxidation, anti-inflammatory and anti-atherosclerotic. Furthermore, previous studiesreported that TSG not only protect against cerebral ischemia through JNK pathways andinhibition of intracellular ROS generation but also antagonizes age-related α-synucleinoverexpression in the hippocampus of APP transgenic mouse model of Alzheimer’s disease.More importantly, TSG protects human neuroblastoma SH-SY5Y cells againstMPP+-induced cytotoxicity.Our recent study showed that TSG not only protect PC12cells from6-OHDA-inducedapoptosis through the ROS-NO pathway but also associated with modulating JNK activation.To investigate further the mechanisms, this study was designed to1) evaluate the putativeneuroprotective properties of TSG in vivo in the MPTP mice model of PD and2) determinewhether the protective mechanisms involve inhibition of the ROS mediated mitochondrialand JNK, P38pathway as suggested by in vitro data.【Objective】1. To PD establish the mouse model.2. To investigate the effects of TSG on the alteration of behavioral，morphological andbiochemical in MPTP induced PD mouse model.3. Determine whether the inhibition of the ROS mediated JNK, P38and mitochondrialpathways are involved in the neuroprotective effects of TSG. 【Methods】1. Animals were all randomly divided into7groups,10for each group：(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.p.);(E) NS (i.p.)+TSG20mg/kg/day(i.g.);(F) NS (i.p.)+TSG40mg/kg/day (i.g.);(G) MPTP (i.p.)+NAC200μM/kg (i.p.).Animals in groups A–G received saline or MPTP (30mg/kg, i.p.) daily for7days,vehicle (saline) or TSG or NAC treatment (i.g.) was started1h after the MPTPadministration and given daily for14days. The pole test and open field test wereperformed one day following treatments with each experimental schedule to measurebehavioral change.2. Mice were anesthetized after behavioral tests. The TH-positive neurons observed byImmunofluorescence histochemistry.3. Detect the level of ROS by automatic fluorescence microplate.4. The level of DA、DOPAC and HVA are detected by HPLC.5. Western Blot detect the expression of JNK, p-JNK, P38, p-P38, ERK, p-ERK, bcl-2, bax,cyt c, smac, cleaved-caspase-9, cleaved-caspase-3, cleaved-caspase-6protein.【Results】1. Behavioral experiment results showed that, following expose to MPTP, TLasignificantly increased,（P＜0.01）compared with control group. After received TSG, adramatic reduction of TLawas observed compared with MPTP group（P＜0.01）.Especially40mg/kg TSG group,（Fig.2a, P <0.01）. TSG only didn’t significantlychange（P＞0.05）. NAC made the TLasignificantly reduced （P＜0.01）. In Open fieldtest，the velocity (V) was less（P＜0.01）, and the time spent in the center (T) wassignificantly increased（Fig.2b, P <0.01）,in the MPTP treated mice as compared to thecontrol mice. While the mice treated with TSG ameliorated this trend in adose-dependent manner, the V recover to4.367cm/s and T decreased to11.00s. Micetreated with TSG alone didn’t show any significant differences compared with control group（P＞0.05）, the mice treated by NAC showed a great improvement in V of mice,and T decreased significantly compared with MPTP treated group（Fig.2c, P <0.05）.2. Other researchers, blinded to the treatment schedule, performed the SNpc neuronalcounts manually. The immunofluorescence histochemistry results showed thatcompared with the controls, MPTP treated group, significantly lower TH positive cells(an approximately66.9%decrease) in the SNpc were observed,（P＜0.01）. And therewas a great positive relationship between the TSG dose and the number of TH positivecells. Compared with control group,TSG alone did not reach significance change（P＞0.05）. Similar to the results obtained from40mg/kgTSG+MPTP group, NAC treatedincreased the TH positive cells significantly (Fig.3, P＜0.05).3. HPLC results showed that striatal DA level was decreased by82%compare to thecontrol group (P <0.01). The levels of DOPAC and HVA in striatal are respectivelydecreased to15%and19%of the control. While the levels of DA, DOPAC, and HVAshowed a tendency to recover gradually after TSG treatment. The affect of high doseTSG was more obvious than low dose. Their level didn’t reach significance changewhen received TSG only（P＞0.05）. As antioxidant, NAC not only increased the levelof DA but also its metabolites significantly compare to MPTP alone treated group（Fig.4, P <0.05）.4. The level of ROS was detected by the Automatic fluorescence microplate：after MPTPinjection，the ROS formation was elevated obviously compare with the control group(P<0.01). TSG (20mg/kg,40mg/kg) or NAC co-administration attenuated MPTPinjection-induced ROS formation, and the effect of TSG is in a dose-dependent manner.TSG single administration did not influence the ROS formation（P＞0.05）. This resultsuggests that TSG might act in a similar manner as NAC（Fig.5）.5. Western blot results showed compared with MPTP treatment groups，that TSGpretreatment attenuated the expression of the phosphorylated JNK and P38in adose-dependent manner compared with the MPTP treated group. But TSG didn’timprove MPTP induced phosphorylated ERK decline. NAC inhibited this increase. 【Conclusion】1. TSG has neuroprotective effects.2. Our results indicated that the protective effects of TSG in the PD mouse model aremediated，at least in part，via the ROS mediated JNK, P38and mitochondrial pathway.