The Effect And Mechanism Research Of Salidroside On The Pathological α-synuclein

Parkinson`disease(PD)is the second most common neurodegenerative disease associated with aging.Bradykinesia,muscle rigidity,postural instability and static tremor are the main clinical manifestations.The main pathological hallmark of PD is the loss of dopaminergic(DA)neurons in the substantia nigra pars compacta(SNc)and the presence of Levy bodies(LBs)composed of aggregated alpha-synuclein(α-syn)in surviving SNc neurons.α-Syn,a presynaptic protein,mainly involved in synaptic plasticity and DA transmission.It`s abnormal aggregation after mutation produces cytotoxicity and eventually leads to cell death.Cell damage resulted from abnormal aggregation of α-syn is the core mechanism of Parkinson’s disease occurs.Current therapy is mainly symptomatic treatment with levodopa preparations.Although,this therapy can alleviate the most movement symptoms of the patients,but long-term use has serious side effects and significant attenuation in curative effect.It can not effectively prevent or delay the progress of PD.Traditional Chinese Medicine(TCM)in the treatment of PD has wealthy experience and methods,however,it`s still facing many problems because of slow treatment effect,unapparent relief in the main symptoms and the unclearly specific mechanism.So the drug research that can interfere PD key pathological mechanism and slow disease progress has been becoming the current neuroscience research emphasis and focus.Salidroside(p-hydroxyphenethyl-β-D-glucoside,Sal),is the main active ingredient of the genus Rhodiola rosea L in crassulaceae family,has a variety of pharmacological effects including anti-oxidative,anti-inflammatory,anti-tumor,and anti-fatigue properties.Our previous studies found Sal pretreatment protected PC12 cells from 1-methyl-4-phenylpyridinium(MPP+)-induced apoptosis via modulating the NO pathway and PI3K/Akt pathway in the MPP+-induced PD vitro model.Sal protected PC12 cells and dopaminergic neurons in mice against MPP+/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced apoptosis through modulating ROS-NO related mitochondrion pathway.In addition,Sal inhibited the excessive accumulation of α-syn.However,the mechanism of Sal inhibiting or removing the pathological α-syn is still unknown.In the normal brains,only 4% of α-syn is composed of the α-syn phosphorylated at serine 129(p Ser129-α-syn).In the pathological condition,approximately 90% of α-syn deposited in LBs is p Ser129-α-syn.Missense point mutations in the SNCA gene that encodes α-syn protein such as A30 P,A53T cause the autosomal dominant early-onset PD.Studies from overexpression of WT/A30P/A53 T α-synuclein transgenic mice showed motor deficits and neurodegenerative alterations,and reduce the abnormal accumulation of α-syn in neurons could inhibit the formation of the neuronal inclusions and the disease progression.The Ubiquitin-proteasome system(UPS)and the Autophagy-lysosome pathway(ALP)are the major two pathways maintaining the proteome homeostasis in all eukaryotic cells.Research suggests that WT/A30P/A53T-α-syn can be degraded by both the UPS and ALP.Some studies shown that UPS is the main degradation pathway for endogenous α-syn and overexpression of α-syn.UPS impairment induced intracellular protein imbalance is considered to be the main cause of PD,Maintaining UPS normal function is considered to be the key target for slowing PD progress and protecting the neurons.In the present study,we induced PD model with Six-Hydroxydopamine(6-OHDA)and WT/A30P-α-syn transfection in SH-SY5 Y cells to investigate whether the Sal could possess the effect of improving the UPS function,inhibiting or removing the pathological p Ser129-α-syn/α-syn and protecting neurons.Objective:(1)Establish 6-OHDA-induced PD model in SH-SY5 Y cells to investigate whether the Sal could possess the effect of protecting the cells viability and inhibiting the p Ser129-α-syn generation and mechanism behind it.(2)Establish WT/A30P-α-syn transfection-induced PD model of α-syn overexpression in SH-SY5 Y cells to investigate whether the Sal could possess the effect of protecting the cells viability and removing WT/A30P-α-syn and mechanism behind it.Methods: MTT assay determine the cell viability,western blot and immunocytochemistry test the p Ser129-α-syn protein level,proteasome activity assay measure the 20 S proteasome activity,plasmid transfection establish the model of α-syn overexpression.Results:(1)6-OHDA treatment decreased the viability in a dose-dependent manner and increased p Ser129-α-syn level,Sal pretreatment significantly improved the cell viability and inhibited the increase of p Ser129-α-syn.6-OHDA(100μM)treatment decreased the 20 S proteasome activity and protein levels of Free ubiquitin,PARKIN,UCH-L1 to 41.0±6.2%,40.4±6.3%,38.9±7.9%,41.6±2.8%(P<0.01)respectively and increased the HMW-ubiquitinated proteins level to 201.5±15.5%(P<0.01).Sal pretreatment increased the 20 S proteasome activity and protein levels of Free ubiquitin,PARKIN,UCH-L1 to 81.0±6.1%(P<0.01),84.9±12.0%(P<0.05),77.6±6.5%(P<0.01),and 83.5±7.4%(P<0.01)respectively.Besides,Sal pretreatment decreased the HMW-ubiquitinated proteins level to 118.8±17.4%(P<0.05).After inhibited the proteasome activity by MG132,the effect of Sal attenuated p Ser129-α-syn was vanished(P<0.01).Inhibited the autophagy with HCQ had no significant influence on pSer129-α-syn level mediated by Sal(P>0.05).Under the autophagy and UPS inhibition conditions,the effect of pSer129-α-syn level reduced by Sal was vanished(P<0.01).(2)SH-SY5 Y cells transfected with WT/A30P-α-syn 24 h,then treated with Sal(40μM)another 24 h,the α-syn protein levels significantly decreased(P<0.01).WT/A30P-α-syn transfection and Sal treatment had no significant influence on protein levels of PARKIN and UCH-L1(P>0.05).Sal treatment after WT/A30P-α-syn transfection attenuated the α-syn protein levels decreased to 35.3±7.4% and 34.0±7.0%(P<0.01)respectively.After inhibited the proteasome activity by MG132,the α-syn protein levels increased to 203.0±14.8% and 218.0±15.1%(P<0.01)respectively.Proteasome activity assay revealed that after SH-SY5 Y cells transfected with WT/A30P-α-syn,the 20 S proteasome activities were decreased to 49.7±4.9% and 44.0±4.0%(P<0.01)respectively,after Sal treatment,the 20 S proteasome activities were recovered to 75.7±6.1%(P<0.01)and 66.7±7.0%(P<0.05)respectively,after Sal and MG132 treatment,the 20 S proteasome activities were decreased to 26.0±2.9% and 26.7±4.7%(P<0.01).MTT assay shown that cells transfected with WT/A30P-α-syn 24 h,the cell viabilities were decreased to 62.0±3.1% and 57.5±2.4%(P<0.01)respectively,after Sal treatment,the cell viabilities were recovered to 75.6±2.3%(P<0.01)and 69.7±4.1%(P<0.05)respectively,after proteasome activity inhibition,the cell viabilities mediated by Sal were decreased to 51.0±5.7% and 43.8±2.5%(P<0.01)respectively.ALP inhibited by HCQ had no significant influence on protein levels of α-syn mediated by Sal(P>0.05).After proteasome activity and ALP inhibition,α-syn protein levels mediated by Sal were increased to 258.7±28.9% and 179.0±11.6%(P<0.01)respectively.Conclusion:(1)Sal attenuated the 6-OHDA induced cytotoxicity,inhibited the p Ser129-α-syn level increase through improving the UPS function.(2)Sal exerted cell protective effect mainly through improving the 20 S proteasome activities and removing the WT/A30P-α-syn.