Neuroprotective Effects Of Salidroside In The MPTP Mouse Model Of Parkinson’s Disease Via The Inhibition Of ROS-NO Pathway

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss ofdopaminergic neurons in the substantia nigra pars compacta（SNpc） and the presence ofα-synuclein deposits in various brain regions. The precise pathogenic mechanisms inPD remain incompletely understood，oxidative stress，mitochondrial dysfunction andthe accumulation of misfolded proteins may play a fundamental role in the development ofPD.1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine（MPTP），a PD-inducing neurotoxinprecursor that crosses the blood-brain barrier and is metabolized/oxidized by glial cellsto1-methyl-4-phenylpyridinium (MPP+). MPP+through dopamine transporters isreleased and taken up by dopaminergic neurons. MPP+inhibits mitochondrialcomplex I，triggering oxidative stress and eventual cell death. The MPTP model inC57BL/6is widely used to study neuroprotective effect of drugs because it recapitulatesthe primary pathological and biochemical features of PD，such as oxidative stress，mitochondrial dysfunction and apoptosis.Current PD medications treat symptoms；none halt or retard dopaminergic neuron degeneration. And long-term use alternative drugs also exist resistance and side effects.Recently，whether plant medicine can prevent or stop the PD progress cause the greatinterest of people.Rhodiola rosea L，a perennial plant of Crassulacea family，has been used forthousands of years in traditional Tibetan medicine to protect against mountain sickness，improve cognitive function and resist aging.Salidroside(p-hydroxyphenethyl-b-D-glucoside，C14H20O7，Sal)，a phenylpropanoid glycosideextracted from Rhodiola rosea L，has been reported to possess various pharmacologicalproperties，including anti-oxidative，anti-aging，anti-cancer and anti-inflammation. Recentstudies showed that sal can inhibit H2O2or Aβ-induced oxidative stress and apoptosisin neuronal pheochromocytoma cells，neuroblastoma cells，and hippocampalneurons. Experiments in vivo also showed ability of sal to reduce thecerebral ischemia-reperfusion injury in rat by its antioxidant activity. Werecently demonstrated that Sal can attenuate MPP+-induced apoptosis in PC12cells bysuppressing ROS accumulation and inhibiting the NO pathway. The aim of this study wasto clarify whether the neuroprotective effects of sal in the MPTP mouse model ofParkinson’s disease and whether involve the inhibition of the ROS-NO pathway assuggested by in vitro data.【Objective】1. Establish the subcute MPTP mouse model of PD.2. Investigate the effects of Sal on the alteration of behavioral，morphology andneurotransmitter in MPTP induced PD mouse model.3. Determine whether the neuroprotective effects of Sal in the MPTP mouse model ofPD and whether involve the inhibition of the ROS-NO pathway.【Methods】1. All animals were were randomly divided into6group：saline control group（a），MPTP group（b），MPTP+Sal（15mg/kg）group(c)，MPTP+Sal（45mg/kg）group(d)，Sal（15mg/kg）group(e) and Sal（45mg/kg）group(f). Mice received dailyintraperitoneal (i.p.) injections of vehicle (saline) or MPTP (30mg/kg/day) dissolved in physiological saline for five consecutive days to induce Parkinsonism.The behavioral testswere tested in the second week.2. The second day after behavioral tests， mice were anesthetized.Immunofluorescence histochemistry observe the TH-positive cells and DAT-positiveneurons.3. The level of ROS and NO was detecd by automatic fluorescence microplate.4. The level of3-NT was detected by ELISA.5. Western Blot detect the expression of iNOS and nNOS protein.6. The level of MDA and the activity of GSH、GSH-Px、SOD were detecd byautomatic fluorescence microplate.7.The level of DA、DOPAC and5-HT are detected by HPLC.【Results】1. Behavioral experiment results showed that，the climbing pole test showed that thetime of climbing pole of the mouse in MPTP group was significantly prolonged，comparedwith the saline control group（P﹤0.01）. After the administration of different levels ofsal，the time of climbing pole was prolonged，by a manner of does-dependent.（P﹤0.05，P﹤0.01）. The results of griding test were opposite with the results of climbing poletest，the time of griding of the mouse in MPTP group was significantly reduced comparingwith the saline control group. And after the different level of Sal was injected，the time ofswimming of the mouse was prolonged（P﹤0.05，P﹤0.01）.Simple Sal group comparedwith the control group was not statistically significant.2. The immunofluorescence histochemistry results showed that numbers ofTH-positive cells and DAT-positive neurons in the substantia nigra were significantlyreduced in MPTP group comparing with the saline control group（P <0.01），but after thedifferent levels of sal were injected，decreasing of TH-positive cells and DAT-positiveneurons were partially reversed（P﹤0.05，P﹤0.01）Simple Sal group compared with thecontrol group was not statistically significant.3. The level of ROS and NO was detected by the Automatic fluorescence microplate：the levels of ROS increased to(269.3±2.7)%(P<0.01) in MPTP group，comparing with levels of (107.4±2.1)%in the normal control group，as well as the level of ROS dropsrespectively for (224.6±3.3)%(P<0.05)，(154.7±3.4)%(P<0.01) in15，45mg/kg Salpretreatment group. The levels of NO increased to (179.6±3.3)%(P<0.01)in MPTPgroup，compared with the NO level (99.3±2.9)%in the normal control group，as well asthe level of NO drops respectively to(154.1±3.7)%(P<0.05)，(133.1±4.1)%(P<0.01) in15，45mg/kg Sal pretreatment group. Simple Sal group compared with the control groupwas not statistically significant.4. The detected results of3-NT shows that compared with the3-NT level(0.7±1.1)%in the normal control group，the levels of3-NT increased to (3.1±1.7)%(P<0.01) in MPTP group，and the level of3-NT respectively reduced to(2.7±1.6)%(P<0.05)，(1.5±2.4)%(P<0.01) in15，45mg/kg Sal pretreatment group. Simple Sal groupcompared with the control group was not statistically significant.5. Results of Western blot indicate that compared with MPTP treatment groups，theexpressive level of nNOS and iNOS reduced in turns.6. The detected results of MDA shows that compared with MPTP treatmentgroups，the expressive level of MDA reduced in turns.The detected results of GSH、GSH-Px and SOD shows that compared with MPTPtreatment groups，the expressive level of GSH and the activity of GSH-Px and SODincreased in turns.7. HPLC results showed that the level of DA、DOPAC and5-HT was significantlyreduced comparing with the saline control group（P﹤0.01）. And after the different levelof Sal was injected，the level increased by a manner of does-dependent（P﹤0.05，P﹤0.01）.【Conclusion】1. Sal has neuroprotective effects in the MPTP mouse model of PD.2. Our results lead to the hypothesis that the protective effects of salidroside in themouse are mediated，at least in part，via the ROS-NO pathway.