Experimental Studies On The Neuroprotective Effects Of Isradipine On PC-12 Cells Apoptosis Induce By Proteasome Inhibitor

Parkinson's disease (PD) is a neurodegenatative disease. The incidence rate and prevalence of PD will increase with age, and the environmental pollution will increase the incidence. PD is characterized by the degeneration and loss of dopaminergic neurons in the brain's substantia nigra pars compacta region. The dopamine is decrease, and the Ach are correspondence accentuation. The static tremor, rigidity, and bradykinesia are the major clinical manifestation of PD, is considered as the most frequently movement disorders in the world. The main treatment of PD is the replacement therapy using levodopa, it can not slow down the progress of PD. So it is necessary to find some ways to protect the dopaminergic neurons from death and slow down the progress of PD.Object: study the neuroprotective effects of isradipine on dopaminergic neurons (PC12 cells) in Parkinson cell model induce by lactacystin a proteasome inhibitor. And it will explore a new way on the neuroprotective treatment of PD.Methods: Induce the cell damage of PC12cell by lactacystin a selective proteasome inhibitor to establish Parkinson's cell model, and it will be pretreated with isradipine. Cell survival rate are detect by MTT assay, Flow cytometry and TUNEL assay to detect the apoptosis, and the change of positive cells of TH and Alpha Synuclein are detected by in immunoctochemical stain, The [Ca2+]i detect by Flow cytometry use fluo-4/AM. Results:1. Establish a PD cell model.survival rate in difference concentration of lactacystin(5,10,15,20μmol/L) is 78.24%±12.35%,63.88%±11.80%,60.18%±8.95%,58.26%±9.56% .it shows that the survival rate will decrease by the increase of concentration of lactacystin. In lactacystin group, TH positive cells are 5.06±2.18 significantly decrease compare with control group38.86±7.40 (p<0.01); Alpha Synuclein positive cells are 26.39±1.93 significantly increase compare with control group(p<0.01). In lactacystin group, apoptosis ratio is 11.21%, significantly increase compare with control group0.88%±0.18% (p<0.01); positive cell of TUNEL is 80.89±13.47 significantly increase compare with control group5.92±3.16(p<0.01).2. Neuroprotective effects of isradipine on PC-12 cells apoptosissurvival rate in difference concentration of isradipine (5,10,20umol/L) pretreatment group is 64.32%±7.98%,70.66%±9.95%,75.02%±8.63%, in 20umol/L isradipine group significantly increase compare with lactacystin group, significantly decrease compare with the control group.The positive cell of TH in isradipine(20umol/L) pretreatment group is17.00±4.37 significantly increase compare with the lactacystin group; The positive cell of Alpha Synuclein in isradipin(e20umol/L) pretreatment group is18.58±4.43 significantly decrease compare with the lactacystin group.The apoptosis ratio of isradipine(20umol/L) pretreatment group is 3.15%±1.61% significantly decrease compare with the lactacystin group, and there re no significantly difference with control group. The positive cell of TUNEL in isradipine(20umol/L) pretreatment group is18.14±7.86 significantly decrease compare with the lactacystin group, and is significantly increase compare to control group.3. Calcium imaging by Flut-4/AM and Flow cytometryThe [Ca2+]i are significantly decreases in the lactacystin group compare to control group, and it is getting more and lower along with the time in 24hours; compare to the lactacystin group [Ca2+] i are lower in isradipine(20umol/L) pretreatment group in the first 1hour, but the difference is not significantly; And after 1 hour the [Ca2+] i Upswing, in 24hour after the treatment the [Ca2+] i become normal again. [Ca2+] i in the simply isradipine(20umol/L)are there are no significantly difference, compare to control group.Conclusion:1. Lactacystin can induce the apoptosis in PC-12 cells.2. Lactacystin can decrease the [Ca2+] i in PC-12 cells.3. Isradipine can inhibit the apoptosis induce by lactacystin in PC-12 cells.