Study On Isradipine Prevents MPP~+-Induced Damage And Molecular Mechanisms In Human Neuroblastoma SH-SY5Y Cells

Parkinson’s Disease is a kind of degenerative diseases of the central nervous system characterized by a significant reduction in striatal dopamine content and the progressive deletions of the nigrostriatal dopaminergic neurons.The main clinical features of PD are bradykinesia,increased muscle tone and resting tremor etc.Parkinson’s disease is a major cause of neurological disability in the adult population,the incidence of Parkinson’s disease in the elderly population is second only to Alzheimer’s disease, the second most neurodegenerative diseases.According to statistics, in 2030, the world population over the age of 50, there will be 930 million people in distress[1,2].Currently, there is no effective treatment and cure of drug research for its pathogenesis, for Parkinson’s disease leads to the specific mechanism of dopaminergic neurons preferentially lost is not very clear.This study apply1-methyl-4-phenyl pyridine ion(1-methyl-4-phenyl pyridine, MPP+) to establish an in vitro model of Parkinson’s disease in SH-SY5 Y cells, and then investigate Isradipine inhibit MPP+-induced intracellular Ca2+increase and mitochondria damage,and thus intervent MPP+-induced SH-SY5 Y autophagy and play the role of intervention in vitro model of Parkinson’s disease.Objective: To study the protection of Isradipine on MPP+-induced damage in SH-SY5 Y cells and to investigate the protective mechanism.Methods:MTT(methyl thiazolyl tetrazolium)assay is used to detect cell vitality rate after treatment with MPP+and Isr pretreatment with MPP+for different time;Flow cytometry analysis intracellular reactive oxygen species(reactive oxygen species,ROS) level and the change of mitochondrial membrane potential treatment with MPP+;Fluo-3/AM staining was used to determine intracellular Ca2+fluorescence and observed under Fluorescence microscope after treated with MPP+ and pretreatment with Isr,and using a microoplate reader record intracellular Ca2+ fluorescence intensity;Dansylcadaverine(monodansylcadaverine,MDC) staining is used to observe autophagic vacuoles under the fluorescence microscope after treatment with MPP+ and pretreatment with Isr following MPP+in SH-SY5 Y cells;Flow cytometry analysis quantitatively the formation of acidic autophagy in the cell by the MDC vital staining; Observe the phenomenon of mitochondria autophagy with Transmission electron microscopy; Western blot analysis the protein expression changes of mitochondrial cytochrome C,P62,and LC3.Results:(1)MTT assay experimental results showed that after pretreatment with Isr,MPP+on the cells of proliferation inhibition was significantly reduced,Isr inhibited MPP+-induced apoptosis of SH-SY5 Y cells in a dose-dependent manner,and the best dose is 2μM Isr.(2)Flow cytometry test results suggested that,compared with model group,Mitochondrial membrane potential levels were rised with JC-1 staining,suggesting that mitochondria damage was reduced.()Flow cytometry test results suggested that,compared with model group,DCFH-DA staining fluorescence intensity was reduced in the Isr group,indicating that ROS levels was decreased;(4)Fluo-3/AM staining results showed that intracellular Ca2+ increased after treated with MPP+,and a certain dose-dependent manner;pretreatment with Isr intracellular Ca2+ intensity decreased.(5)MDC dyeing experiment results found thatthere were a large number of green fluorescent dot gathered marked by MDC in the cells with MPP+treatment while green fluorescent dot aggregation decreased significantly in the cell with drug.(6)Electron microscopy test results found that after MPP+treatment,the nucleus chromatin were condensed,the mitochondria structure was disorder and more vacuoles and its number was decreased significantly,the contents can be seen in the part of cavity,and some mitochondria was wrapped into vesicles;There were no clear vacuoles in the cytoplasm with Isr treatment and cell morphology were close to the normal state.(7)Western blotting test results indicated that compared with control group,the expression of cytochrome C and autophagy related protein P62 increased in MPP+treatment group, the ratio of LC3-Ⅱ/LC3-Ⅰwas rised, but the expression of cytochrome C induced by MPP+was significantly inhibited in SH-SY5 Y cells with drug,reduced the expression of autophagy-related protein P62 and LC3-Ⅱ.Conclusion: MPP+can increase intracelluar Ca2+, induce intracellular ROS accumulation,decrease mitochondrial membrane potential,leading to mitochondrial dysfunction and cell damage.Its mechanism may be related to the expression of activating autophagy protein P62 and LC3; Isr decrease intracelluar Ca2+,canreduce ROS production, improve the capacity of scavenging free radicals and increase mitochondrial membrane potential in SH-SY5 Y cells,additionally,it also can down-regulate the expression of autophagy protein, decrease the level of autophagy,thereby which play a role of neuroprotection,delay the occurrence and development of PD.