Salidroside Protects Against Neuronal Injury Through DJ-1/Nrf2 Antioxidant Pathway

Parkinson’s disease(PD)is the second most common progressive neurodegenerative disorder in the world,affecting more than 1% of population over the age of 60.PD is attributed to loss of dopaminergic neurons in the SNpc and characterized by the presence of ubiquitinated alpha-synuclein(α-syn)containing cytoplasmic inclusions called Lewy bodies in surviving SNpc neurons.Although the accurate mechanism of PD is not known,oxidative injury and associated apoptotic cell death are believed to play an important role.Large quantity of studies has shown that antioxidant intervention is effective for the treatment of PD and other neurodegenerative diseases.At present about the treatment of PD levodopa(levodopa,L-DOPA)has long been known as the "gold standard" drugs,however the only 5-10% into the blood brain barrier,the rest of the metabolism of levodopa for dopamine exists in various parts of the body,causing a series of side effects,therefore,looking for new alternative,small side effects of drugs is imperative.In recent years,it is a hot issue to seek antioxidant drugs from plant source drugs and active ingredients as delaying the pathogenesis of PD.Numerous studies have shown that the intervention of antioxidants is effective in the treatment of PD and other neurodegenerative diseases.Salidroside is a major component extracted from Rhodiola rosea L,which is a plant mainly popular in traditional medicine in Asian and Eastern European countries.It is shown that Sal has many pharmacological activities,including neuroprotective effects,anti-aging and anti-oxidation.Our previous studies have shown that Sal protects against apoptosis in cells by inhibiting the NO and PI3k/Akt pathway.We also show that Sal protects against PD in mouse model through ROS-NO related mitochondrion pathway.These results suggest that the antioxidant activity of Sal plays a pivotal role in the neuroprotective effects against PD.However,the mechanism underlying the neuroprotective effects of Sal is still far from being completely understood.The neurotoxin MPTP(1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine)causes neurotoxicity through the active metabolite MPP+(1-methyl-4-phenylpyridinium).Moreover,oxidative stress is involved in MPP+-associated PD-like neuronal injury.MPP+could be selectively taken up by dopaminergic neurons via the dopamine transporter of the plasma membrane.MPP+results in neuronal loss in substantia nigra and striatal dopamine(DA)depletion,leading to PD-like behavioral impairments.MPP+/MPTP induced neuronal injury in neuron cells and neurodegenerative disorder in animals are usually used for the study of the pathogenesis and treatment of PD.The relationship between DJ-1(PARK7)and PD is first proposed by Bonifati and his colleagues,its abnormal regulation was closely related to the development of PD,and its genetic variation could lead to the autosomal recessive early onset PD.DJ-1 is a new type of antioxidants,are present in the nerve and peripheral tissue,located in the nucleus and cytoplasm,involved in apoptosis,protein folding,with activity and transcription regulation.DJ-1 can reduce the cellular damage caused by oxidative stress in various ways and improve the movement disorder caused by neurotoxin.Many documents show that Nrf2 cut is associated with the pathogenesis of PD,and raised the Nrf2 can suppress the PD process,a certain degree of and Nrf2 can by activating stage II antioxidant enzymes play an important nerve protective effect.Therefore,more and more attention has been paid to the strategy of reducing oxidative stress in the treatment of PD by studying DJ-1/Nrf2 as the target.In the present study,SH-SY5 Y cells and mice were used to establish PD nerve injury model to investigate whether Sal could protect the apoptosis of dopamine neurons by regulating the DJ-1 /Nrf2 pathway.Part one :Protective effect of Sal on MPP+-PD model in SH-SY5 Y cellsObjective:Establish cell model of MPP+-induced PD,The effective protection concentration of MPP+induced cell apoptosis and Sal’s neuroprotection effect against MPP+in SH-SY5 Y cells were selected.The protein and m RNA expression level of Nrf2,SOD1,SOD2,GCLc and DJ-1 was measured.Methods:1)SH-SY5 Y cells were exposed to 0–600 μM MPP+for 12–48 h to select the effective protection concentration Sal-exhibited MPP+.2)Using MTT detection method to detect the influence of Sal on the proliferation of SH-SY5 Y cells.3)Cells were stained with Hoechst to observe the morphological changes of nuclei.Cells death was also determined using Annexin V/PI assay kit and analyzed by flow cytometry.4)To examine the mechanism of Sal-exhibited neuroprotective effects,we examined the effect of Sal on the protein expression of key antioxidant proteins,including Nrf2,SOD1,SOD2,GCLc and DJ-1.5)To examine the mechanism of Sal-exhibited neuroprotective effects,we examined the effect of Sal on the m RNA expression of key antioxidant proteins,including Nrf2,SOD1,SOD2,GCLc and DJ-1.Results:1)Cells were exposed to 0-600μM MPP+for 12-48 h and the results showed that MPP+resulted in a significant decrease of cell viability in a concentration and time-dependent manner.Cells were pretreated with 25-100 μM Sal for 24 h and thenexposed to 500 μM MPP+for an additional 24 h.We showed that Sal concentration-dependently prevented MPP+-induced decrease of cell viability(P<0.05).2)Hoechst stain showed that different concentrations of Sal notably inhibited MPP+induced increase of chromatin condensation,intense fluorescence,and nuclear fragmentation in SH-SY5 Y cells(P<0.05).3)Cell apoptosis was signifiantly increased after 500 μM MMP+for 24 hours.The pretreated Sal could significantly decrease cell apoptosis in a concentration dependent manner(P<0.05).4)The results showed that MPP+induced a significant decrease of the protein expression of DJ-1,Nrf2,GCLc,SOD1 and SOD2(P<0.05).The treatment of Sal significantly prevented MPP+-induced decrease of the protein expression of DJ-1,Nrf2,GCLc,SOD1 and SOD2 of SH-SY5 Y cells in a dose-dependent manner(P<0.05).5)The results showed that 500 μM MPP+induced a significant decrease of the m RNA expression of DJ-1,Nrf2,GCLc,SOD1 and SOD2.The pre-treatment of Sal significantly prevented MPP+-induced decrease of the m RNA expression of DJ-1,Nrf2,GCLc,SOD1 and SOD2 of SH-SY5 Y cells in a dose-dependent manner(P<0.05).Conclusion:These results indicated that Sal protected against MPP+-induced cytotoxicity,cell apoptosis and upregulated protein and m RNA level of DJ-1,Nrf2,GCLc,SOD1,and SOD2 in cells.Part two:Upregulation of Nrf2 is involved in the neuroprotective effects of Sal against MPP+ cytotoxicityObjective:SH-SY5 Y cells were further transfected with si Nrf2 to examine whether the regulation of Nrf2 was involved in the neuroprotective effects of Sal against MPP+cytotoxicity.Methods:1)To examine whether regulation of Nrf2 was involved in the neuroprotective effects of Sal against MPP+cytotoxicity,SH-SY5 Y cells were transfected with si Nrf2.a 、 Control+scramble si RNA;b 、 500μM MPP+scramble si RNA;c 、 500μM MPP++100μM Sal+scramble si RNA;d、500μM MPP++100μM Sal+si Nrf2.2)Cells death was determined using Annexin V/PI assay kit and analyzed by flow cytometry.Cells were also stained with Hoechst to observe the morphological changes of nuclei.3)To examine the mechanism of Sal-exhibited neuroprotective effects,we examined the effect of Sal on the protein expression of key antioxidant proteins,including Nrf2,SOD1,SOD2,GCLc,and DJ-1.4)To examine the mechanism of Sal-exhibited neuroprotective effects,we examined the effect of Sal on the m RNA expression of key antioxidant proteins,including Nrf2,SOD1,SOD2,GCLc,and DJ-1.5)ROS level was determined using DCFH-DA staining.GSH level was determined by an assay kit.Results:1)Sal-induced decrease of chromatin condensation,intense fluorescence,and nuclear fragmentation in MPP+-treated cells were inhibited by si Nrf2(P<0.05).2)Silencing of Nrf2 significantly suppressed the decrease of Annexin V/PI-stained cells induced by Sal in MPP+-treated SH-SY5 Y cells(P<0.05).3)Silencing of Nrf2 significantly inhibited Sal-induced increase in protein expression of Nrf2,GCLc,SOD1,and SOD2 in MPP+-treated SH-SY5 Y cells.However,Nrf2 silence did not significantly affect Sal-exhibited effects on DJ-1 expression(P<0.05).4)Silencing of Nrf2 significantly inhibited Sal-induced increase in m RNA expression of Nrf2,GCLc,SOD1,and SOD2 in MPP+-treated SH-SY5 Y cells.However,Nrf2 silence did not significantly affect Sal-exhibited effects on DJ-1 expression(P<0.05).5)Sal inhibited MPP+-induced increase of reactive oxygen species(ROS)and decrease of glutathione(GSH)level.Silencing of Nrf2 significantly inhibited Sal-induced decrease of ROS level and increase of GSH level in MPP+-treated cell(P<0.05).Conclusion:The results indicated that Nrf2-regulated antioxidant enzymes were involved in the neuroprotective effects of Sal against MPP+toxicity.However,the silence of Nrf2 did not significantly affect Sal-exhibited effects on DJ-1 expression.Part three :Upregulation of DJ-1 is involved in the neuroprotective effects of Sal against MPP+ cytotoxicityObjective:SH-SY5 Y cells were further transfected with si DJ-1 to examine whether regulation of DJ-1 was involved in the neuroprotective effects of Sal against MPP+cytotoxicity.Methods:1)To examine whether regulation of DJ-1 was involved in the neuroprotective effects of Sal against MPP+cytotoxicity,SH-SY5 Y cells were transfected with si DJ-1.a 、 Control+scramble si RNA;b 、 500μM MPP++scramble si RNA;c 、 500μM MPP++100μM Sal+scramble si RNA;d、500μM MPP++100μM Sal+si DJ-1.2)Cells were stained with Hoechst to observe the morphological changes of nuclei.Cells death was also determined using Annexin V/PI assay kit and analyzed by flow cytometry.3)To examine the mechanism of Sal-exhibited neuroprotective effects,we examined the effect of Sal on the protein expression of key antioxidant proteins,including Nrf2,SOD1,SOD2,GCLc,and DJ-1.4)To examine the mechanism of Sal-exhibited neuroprotective effects,we examined the effect of Sal on the m RNA expression of key antioxidant proteins,including Nrf2,SOD1,SOD2,GCLc,and DJ-1.5)ROS level was determined using DCFH-DA staining.GSH level was determined byan assay kit.Results:1)Sal-induced decrease of chromatin condensation,intense fluorescence,and nuclear fragmentation in MPP+-treated cells were inhibited by si DJ-1(P<0.05).2)Silencing of DJ-1 significantly suppressed the decrease of Annexin V/PI-stained cells induced by Sal in MPP+-treated SHSY5 Y cells(P<0.05).3)Silencing of DJ-1 significantly inhibited Sal-induced increase in protein expression of Nrf2,GCLc,SOD1,SOD2 and DJ-1 in MPP+-treated SH-SY5 Y cells(P<0.05).4)Silencing of DJ-1 significantly inhibited Sal-induced increase in m RNA expression of Nrf2,GCLc,SOD1,SOD2 and DJ-1 in MPP+-treated SH-SY5 Y cells(P<0.05).5)Silencing of DJ-1 significantly inhibited Sal induced decrease of ROS level and increase of GSH level in MPP+-treated cells.Silencing of Nrf2 significantly inhibited Sal-induced decrease of ROS level and increase of GSH level in MPP+-treated cell(P<0.05).Conclusion:The results indicated that DJ-1-regulated antioxidant enzymes were involved in the neuroprotective effects of Sal against MPP+toxicity.Morever,the silence of DJ-1significantly affect the expression of Nrf2.Part four :Neuroprotective effects of Salidroside in the MPTP mouse model of Parkinson’s disease through DJ-1/Nrf2 antioxidant pathwayObjective:To prepare MPTP induced PD mouse model,observe the effect of Sal on the behavior and neurohistology of MPTP induced PD mouse model,and explore whether Sal can exert neuroprotective effect on MPTP-PD mice through DJ-1/Nrf2 pathway.Methods:1)Animal grouping and model preparation,experimental mice were randomly dividedinto four groups: A.control group(littermates): intraperitoneal injection of Saline0.1ml/,1 /d,continuous 14d;b.MPTP treated mice PD model group: intraperitoneal injection of MPTP 30mg/kg,1 /d,continuous 7d,after injection of the same volume of Saline 1,/d,continuous 7d;c.Sal treatment group: PD model is first given to Sal45mg/kg,1h after intraperitoneal injection of MPTP 30mg/kg,1 /d,continuous 7d,then only 7d injection of Sal 45mg/kg.D.Sal intervention group: intraperitoneal injection of Sal45mg/kg,1 times /d,continuous 14 d.2)After the end of the experimental design,the pole test and grid test were used to observe the behavioral changes in mice.3)On the 14 day after administration,10 mice were randomly selected from each group to make frozen sections of brain.Immunofluorescence histochemical method was used to observe the effect of Sal on the number of tyrosine hydroxylase(TH)and dopamine transporter(DAT)positive neurons in SN induced by MPTP in PD mice.4)Western Blot method was used to detect the expression of Nrf2,SOD1,SOD2,GCLc,DJ-1 protein in the striatum of mice respectively.Results:1)In mice after administration of MPTP,the gradual emergence of movement disorder symptoms: hypokinesia,limb tremor,arch,instability of gait changes,with the dose increase in the number of days,the symptoms gradually worsened.After the treatment of Sal,it was obviously improved.The main performance was: in the climbing pole experiment,the time of climbing pole of the model group was significantly prolonged(P<0.01).After treatment,the climbing time was shortened(P<0.05).In the suspension experiment,the suspension time of the model group was significantly reduced(P<0.01).After treatment with Sal,the suspension capacity was significantly improved(P<0.05).2)TH and DAT positive cells were mainly distributed in SNpc,and immunofluorescence double labeling showed that TH and DAT coexisted.Compared with the control group,the number of TH and DAT positive cells in the MPTPtreatment group decreased(P<0.05).Compared with the MPTP treatment group,the number of TH and DAT positive cells in Sal intervention group increased significantly(P<0.05).The results showed that the MPTP treatment could induce a decrease in the number of TH and DAT positive cells,while the Sal intervention was protective.3)Compared with the control group,the MPTP damage group mice was significantly reduced in DJ-1,Nrf2,GCLc,SOD1 and SOD2(P<0.05).Sal intervention group compared with control group,the DJ-1,Nrf2,GCLc,SOD1,SOD2 expression level had no significant difference(P > 0.05),but compared with MPTP injury group,DJ-1,Nrf2,GCLc,SOD1 and SOD2 expression ratio significantly inhibited(P <0.05).Conclusion:Sal can improve MPTP-induced-PD mice model of abnormal behavior,reduce the MPTP of the substantia nigra dopaminergic neuron injury in mice,Sal protects MPTP-induced-PD mice model neuroprotection mechanisms may be involved in the DJ-1/Nrf2 pathway.