| Parkinson’s disease (PD) was first described by James Parkinson in1817(Parkinson,1817) and is the second most common neurodegenerative disorder after Alzheimer’sdisease (AD). PD affects approximately2%of the population aged65or over。The loss ofDAergic neurons is progressive, and by the time PD symptoms appear at least50%of allnigral neurons have degenerated, which is accompanied by80%depletion in striatal DAlevels. Even though the etiology of PD remains unclear, several lines of evidence stronglysuggest that oxidative stress and mitochondrial dysfunction play an important role in theneurodegenerative process of this disease. So it is suggesting that compounds interferingboth with reactive oxygen species (ROS) and nitric oxide (NO) production and withimpaired mitochondrial complex I activity might be protective.MPTP is a neurotoxin that produces a parkinsonian syndrome in both humans andexperimental animals. Its neurotoxic effects also appear to involve energy depletion andfree radical generation. MPTP is converted to its metabolite MPP+by MAO-B. MPP+isselectively accumulated by high affinity dopamine transporters and taken up into themitochondria of dopaminergic neurons, where it disrupts oxidative phosphorylation by inhibiting complex I of the mitochondrial electron transport chain. thereby exhibitingdopaminergic neurotoxicity.MPTP or MPP+are widely used and are recognized a classicmodels of PD in the academic world.Rhodiola rosea L, belonging to the crassulaceae plants of the genus rhodiola rosea, isa popular plant in folk medical system in Asian and Eastern European countries. Rhodiolarosea L., contains more than20ingredients including organic acids, flavonoids, tanninsand phenolic glycosides. The plant has a range of pharmacological properties, includinganti-aging, anti-oxidative, anti-inflammatory, anti-fatigue and anti-depressant activities。Salidroside,(p-hydroxyphenethyl-b-D-glucoside; Sal; C14H20O7), which is extracted from Rhodiola roseaL., has been reported to have many pharmacological effects, including antioxidative, anti-aging, andneuroprotective properties.Our previour research demonstrated that Sal protected PC12cells against apoptosisinduced by MPP+. When we investigated further the mechanisms underlying thisphenomenon, we showed that the protective action was achieved through inhibition of theROS-NO pathway, which is known to be involved in MPP+-induced cell death. Thepresent study was designed to investigated whether this protection is mediated byinhibiting the ROS-NO related mitochondrion pathway in vitro and in vivo.Objective:1. Establish MPP+-induced PD cell model, the concentration of MPP+and Salneuroprotection in PC12cells.2. Establish the effect of Sal on the apoptosis of MPP+-induced PC12cells, discussionthe mechanisms of Sal on MPP+-induced apoptosis in PC12cells.3. Establish PD mouse model, investigate the effects of Sal on behavioral，morphology.4. Determine the neuroprotection of sal through ROS-NO Related MitochondrionPathwayMethods:1. PC12cells were divided into control group,100,200,300,400,500,600,700μmol/LMPP+groups for24h to investigate the neurotoxicity of MPP+. Therefore, the treatment of500μM MPP+for24h was used to induce PC12cell injury in subsequentexperiments. Set on normal group, Sal alone group, MPP+group and the differentconcentrations of Sal pretreatmen（t10,50,100μmol/L）groups, cell viability measuredby MTT, Hoechst33258and flow cytometry. Intracellular ROS and NO were detectedby the Automatic fluorescence microplate. The expression of Bcl-2, Bax, Cytochrom-c,Smac, Caspase-3,-6,-9, α-syn in PC12cells was detected by the Western Blotmethod.2. All animals were divided into5group (each group10mice)：control group(intraperitoneal injections of saline solution for14days), Sal treatment group alone(Sal45mg/kg for14days), MPTP treatment group alone (MPTP30mg/kg for7days+equivalent volume of saline solution for7days), and pretreated with differentconcentrations of Sal (MPTP30mg/kg for7days+Sal15or45mg/kg for14days).The behavioral tests were tested in the second week. After behavioral tests,useing immunofluorescence histochemistry to observe the Effect of Sal onMPTP-induced behavioral impairments and loss of DA neurons Measurement of ROSand NO in substantia nigra by automatic fluorescence microplate. Western Blot detectthe expression of Bcl-2, Bax, Cytochrom-c, Smac, Caspase-3,-6,-9, α-syn insubstantia nigra.Result:1. In the study, MTT was used to detect the viability of the PC12cell. Treated with500μM MPP+for24h, cell viability was significantly decreased (53.7±1.7%) comparedwith the control group (P <0.01). when the cells were incubated with differentconcentrations of Sal (10,50，and100μM) for24h prior to MPP+exposure.Compared with the control group, the cell viability recovered to61.1±1.7%(P <0.05),73.5±1.4%(P <0.05), and85.1±2.1%(P <0.01), respectively.2. Hoechst33258staining was employed. The nuclei of normal cells showed ahomogeneous and diffuse staining, with regular contours and rounded shapes. Aftertreatment with500μM MPP+alone for24h, dramatically morphological changes in chromatin morphology, such as crenation, condensation, and fragmentation, wereobserved in PC12cells. However, the number of condensed nuclei was significantlyimproved in the groups pretreated with10,50, and100μM Sal, respectively. Sal alonetreatment had no obvious effect on the cell morphology.3. An annexin V and PI double-stain were used to detect cell apoptosis. After incubationwith MPP+for24h, the percentage of apoptotic cells was significantly increased from2.56±0.23%(control group) to47.54±1.23%(P <0.01). Moreover, the ratio of cellapoptosis were dropped to32.13±1.75%,25.32±2.21%, and10.36±1.12%after10,50, and100μM sal pretreatment, respectively. Sal alone treatment also did notshow any obvious effect (2.87±0.14%).4. The Automatic fluorescence microplate was used for detecting intracellular ROS andNO, exposure of PC12cells to500μM MPP+for24h led to a significant increase inROS/NO compared with the control group (P<0.01). Such accumulation in the level ofintracellular ROS/NO was significantly reduced in a dose-dependent manner bypretreatment of the cells with Sal. Compared with the untreated group, ROS/NOspikes were decreased to3.7/3.2fold,2.3/2.1fold, and1.5/1.4fold at10,50, and100μM Sal, respectively.5. Western Blot results show that, compared with500μM MPP+treated group, Salpretreatment can improve the ratio of Bcl-2/Bax, reduce Cytochrome-c, Smac release,inhibition of Caspase-3,-6,-9activity and α-syn aggregation. The Sal treatment alonehad no significant effect on the expression of these proteins in cells6. The pole test results showed that, compared with the control group, MPTP decreasedmice motor coordination in model group, the mice showed the time to climed down tothe floor (TLA) and The time that it turned completely downward (Tturn) weresignificantly increased (P <0.01). After different doses of Sal, motor coordination inmice significantly improved by a dose-dependent manner (P <0.05, P <0.01).Immunofluorescence staining showed that: Compared with the control group, thenumber of TH-positive cells significantly reduced in MPTP treted group (P <0.01), andpretreatment with Sal15mg/kg or45mg/kg group, TH-positive cells was significantly higher than the MPTP-treated group (P <0.05, P <0.01). In addition, compared withthe control group, The Sal treatment alone had no significant effect on the number ofTH-positive in substantia nigra.7. The Automatic fluorescence microplate showed that: MPTP induced a rapid rise insubstantia nigra ROS/NO levels, approximately1.4/1.3-fold, compared with thecontrol group (P <0.01). However, Sal significantly inhibited MPTP-inducedROS/NO increase. The Sal treatment alone had no significant effect on the expressionof ROS/NO in substantia nigra.8. Western Blot results showed that: compared with30mg/kg MPTP-treated group, salpretreatment can significantly improve the ratio of Bcl-2/Bax, reduce Cytochrome-c,Smac release, inhibition of Caspase-3,-6,-9activity and α-syn aggregation. The Saltreatment alone had no significant effect on the expression of these proteins insubstantia nigraConclusion:1. Sal partially protects DA neurons from MPTP/MPP+-induced apoptosis both in vitroand in vivo2. The mechanisms of Sal protective effects may be through ROS-NO relatedmitochondrion pathway in the MPTP/MPP+-induced cellular damage. |
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