Salidroside Protects Against MPP~+-induced Apoptosis In PC12 Cells By Inhibiting The No Pathway

Parkinson's disease is a common, chronic neurodegenerative disease of the central nervous system that results in progressive degeneration of dopaminergic neurons in the substantia nigra, leading to a massive depletion of the dopamine neurotransmitter in the striatum. Although the etiology of PD remains unclear, several risk factors have been proposed in association with the etiology of the disease, including genetic,environmental factors, oxidative stress, mitochondrial dysfunction, immunization factor and cell apoptosis.1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine(MPTP), is a kind of synthetic drugs derived from the intermediate product which induced Parkinson's syndrome of a variety of animals by a similar neurotoxin and widely used animal model of PD. MPTP, it'self no toxic to the dopamine neurons, But MPTP is converted into 1-methyl-4-phenyl-2, 3-dihydropyridinium(MPP+) by monoamine oxidase-B. MPP+ is selectively taken up by dopaminergic neurons via the dopamine transporter of the plasma membrane. It directly and/or indirectly inhibits mitochondrial complex I, causing abnormal energy metabolism and increased oxidative stress. In addition, MPP+ produces neuronal loss in substantia nigra, striatal dopamine (DA) depletion and behavioral impairments in humans, primates and mice. It also causes a severe Parkinsonian-like syndrome with loss of dopaminergic cells. Of the sources of oxidative stress, NO plays a major role in MPTP-induced models of Parkinson's disease.Salidroside, which is extracted from Rhodiola rosea L., has been reported to have many pharmacological effects, including antioxidative, anti-aging, anti-cancer and, more importantly here, neuroprotective properties. For example, salidroside has been shown to protect against neuronal cell death induced by glutamate and hypoxia/hypoglycemia, and against mitochondrial dysfunction induced by sodium azide. However, whether salidroside can provide protection against dopaminergic neuron injury induced by MPP+ remains unknown. The purpose of this study is to establish a cell model of PD and observe whether salidroside have a protective effect on MPP-induced PC12 cell apoptosis, We discussed the specific protection mechanisms by detecting intracellular NO, NOS, MMP and Ca2+.Aims and contentsAims:Established the model of PD about PC12 cells induced by MPP+, to study whether and how Salidroside protect PC12 cells from MPP+ induced apoptosis by measuring a series of biochemical indicators, provide ideas for the prevention and treatment of PD.Contents:1. We established an in vito model induced by MPP+ in PC12 cells2. Qualitative and quantitative detection of of apoptosis in each group3. Screening the effective concentration of salidroside against MPP+ induced.4. The possible effect of Salidroside was investigated and the related mechanism was probed.Methods1. PC12 cells were prepared and incubated by MPP+ , and were divided into five groups control group, MPP+ group, Salidroside group, MPP++different concentrations of salidroside group.2. MTT assay was used to detect the cell viability, Hoechst 33258 staining was employed to observe morphological changes of the cell nuclear,FCM was used to detect the apoptosis ratio.3. NO, MMP, Ca2+, ONOO- and NOS were detected.4. L-NMMA + MPP+ group, MTT assay was used to detect the cell viability.Results1. PC12 cells were exposed to(100-600 mol/L)concentrations of MPP+ for 12, 24, 36,and 48 h, there was a dose-and time-dependent decrease in cell viability as measured by MTT assay. PC12 cells were treated with 500 mol/L MPP+ for 24h, cell viability was reaching 53±1.5% compared with control group. So we chosed 500 mol/L MPP+ for 24 as control group.2. Compare with MPP+ group, 10, 50, 100 M Salidroside respectively played a protective role against MPP+ induced PC12. These changes in the nuclear characteristics of apoptosis were rescued significantly in the cells pretreated with salidroside, flow cytometric analysis showed that levels of apoptosis were significantly reduced.3. Western blot showed that the expression of nNOS and iNOS increased with the MPP+-induced apoptosis of PC12 cells4. Flow cytometric analysis showed NO and Ca2+, increased and MMP decreased with the MPP+-induced apoptosis of PC12 cells. ELISA methods showed that Salidroside inhibits the MPP+-induced elevation in protein-bound 3-NT.5. Pretreatment with L-NMMA significantly inhibited MPP+ induced apoptosis in PC12 cells compared with the MPP+ alone.Conclusion1. There was a dose- and time-dependent decrease in cell viability following MPP+ exposure.2. PC12 cells pretreatments with different concetration of salidroside significantly decreased levels of MPP+-induced apoptosis, as compared to cells treated with MPP+ alone.3. Salidroside protects against MPP+-induced apoptosis in PC12 cells by inhibiting the NO pathway...