Study On Isradipine Prevents Rotenone Induced Senescence And Molecular Mechanisms In Human Neuroblastoma SH-SY5Y Cells

Parkinson’s disease (PD) is a kind of the central nervous system degenerationcharacterized by the loss of dopaminergic neurons in the nigrostriatal area and thesignificant reduction of dopamine content in striatal.The main clinical features of PDare rest tremor, increased muscle tone and bradykinesia etc.,PD is commonly occured inpeople over the age of60. However the occuring of PD is getting younger and youngerrecently.There are about2millions PD patients in China currently, half of the total PDcases over the world, and the number are increased by10thousands each year. Now PDhas became the third serious threat to the physical and mental health of the elderly aftercardiovascular and cerebrovascular diseases and Alzheimer’s disease in China.Thisstudy use vitro Parkinson’s disease model builded by rotenone to study isradipine inhibitrotenone-induced intracellular Ca2+increase and mitochondrial oxidative stress, andthus intervent rotenone-induced SH-SY5Y cell aging and delay the progressing ofParkinson’s disease.Objective: To study isradipine rotenone-induced protective effect of aging SH-SY5Ycells, and to investigate the protective mechanism.Methods: MTT assay to detect cell viability rate after treatment with rotenone andisradipine/EGTA pretreatment with rotenone for different time; Flow cytometryAnnexin V-FITC/PI double staining to detect apoptosis rate after treatment withrotenone SH-SY5Y cells; Western blotting detect the express of caspase-3inSH-SY5Y cells after treatment with rotenone; Fluo-3/AM staining was used todetermine intracellular Ca2+fluorescence and observed under Fluorescence microscopeafter treatment with rotenone and pretreatment with isradipine/EGTA followingrotenone in SH-SY5Y cells, and using a microplate reader record intracellular Ca2+fluorescence intensity; SA-β-gal reagent box to detect intracellular β-gal activity aftertreatment with rotenone and pretreatment with isradipine/EGTA following rotenone in SH-SY5Ycells;Lipofuscin Elisa kit to detecte intracellular lipofuscin content aftertreatment with rotenone and pretreatment with isradipine/EGTA following rotenone inSH-SY5Y cells, and transmission electron microscopy to observe lipofuscin formationafter treatment with rotenone;Flow cytometry PI staining to detect cell cycle changesafter treatment with rotenone and pretreatment with isradipine/EGTA followingrotenone in SH-SY5Y cells; Flow cytometry was used to detect intracellular activeoxygen (ROS) after treatment with rotenone and pretreatment with isradipine/EGTAfollowing rotenone; To study rotenone-induced cell aging, and the protectionmechanisms of isradipine, using Western blotting analysis to detecte the express of p53,p16, p21, CDK2, cyclinD1, AKT, p-AKT.Results:(1) MTT method. Experimental results showed that rotenone in a certain period of timeand concentration significantly inhibited proliferation of SH-SY5Y cells, and was aging,dose dependent; using isradipine/EGTA pretreatment, rotenone on the cells of theproliferation inhibition was significantly reduced, and the strongest in48h protectiveeffect.(2)Flow cytometry Annexin V-FITC/PI double staining test results showed that the therotenone apoptosis rate increase, but the apoptosis rate does not increase with time;Western blotting detection cleaveg-caspase3protein expression levels do not increasewith time.(3) Fluo-3/AM staining results showed that intracellular Ca2+increased after treatmentwith rotenone, and a certain dose-dependent manner; pretreatment withisradipine/EGTA intracellular Ca2+fluorescence intensity decreased.(4) SA-β-gal kit detected intracellular β-gal activity increasing after treatment withrotenone; pretreatment with isradipine/EGTA, intracellular β-gal activity was reduced.(5) Elisa test results found that lipofuscin increased treatment with rotenone, and therewas a certain dose-dependent manner; pretreatment with isradipine/EGTA decreased intracellular lipofuscin.(6) Transmission electron microscopy results found that nuclei irregular pyknotic innermitochondrial membrane rupture and a few presents vacuolar degeneration, cellmembraes and organelle membranes fuzzy, and lipofuscin granules material inlysosomes.(7) Flow cytometry results showed the level of ROS increased after treatment withrotenone; but the level of ROS decreased after pretreatment with isradipine/EGTA.(8)PI staining results showed that most of cells blocked in the G0phase treated withrotenone; but G0phase cells decreased and,S/G2/M phase cells increased afterpretreatment with isradipine/EGTA.(9) Western blotting detection results showed that the expression of p53, p16, p21rotenone increased with time after treatment with rotenone for different time, but theexpression of CDK2, cyclinD1, p-AKT protein reduced with time; after pretreatmentwith isradipine/EGTA, the expression of p53, p16, p21reduced and CDK2, cyclin D1,p-AKT increased.Conclusion: Rotenone induced SH-SY5Y cell senscence through intracellular Ca2+increase and mitochondrial oxidative stress, leading to cell death, its mechanism ofaction may be related to activation of p53, of p16, of p21and other related proteins ofexpression, inhibit the expression of cell cycle proteins,such as CDK2, cyclinD1protein;Isradipine/EGTA can inhibit the intracellular Ca2+increase and protect mitochondrialdamage, inhibit the expression of p53, p16, p21protein, activation of p-AKT and cyclinCDK2, cyclinD1protein expression, delaying cell aging process.