Effect of insulin on 1-methyl-4-phenylpyridinium ion-induced apoptosis of PC12 cells

BACKGROUND： Insulin receptor （IR） expression in the substantia nigra of patients with Parkinson disease （PD） is not only significantly lower than that in the substantia nigra of normal persons of the same age, but also significantly lower than that in other regions in brain of himself/herself. It suggests that the abnormal effect of insulin receptor-mediated insulin, as a neurotrophic factor, is very possibly related to the loss of dopaminergic neurons in the substantia nigra and striatum in patients with Parkinson disease. OBJECTIVE ： TO observe the interventional effect of insulin on 1-methyl-4-phenylpyridinium ion （MPP^＋）-induced apoptosis of PC12. DESIGN： Controlled observation SETTINGS： Department of Neurology, Beijing China-Japan Friendship Hospital; Department of Neurology Huashan Hospital Affiliated to Fudan University. MATERIALS： PC12 cells were provided by the Cell Bank, Shanghai Institute of Cell Biology, Chinese Academy of Science. MPP^＋, MTT, HOECHST 33258 （Invitrogen Life Technologies）, reverse transcription-polymerase chain reaction （RT-PCR） reagent （Takara Shuzo Co., Ltd.）, flow cytometer （Bacton Dickionson, San Jose, CA）, enzyme labelling instrument （Bio-Tek, Winooski, VT） and PCR circulation instrument （Takara Shuzo Co., Ltd） were used in this study. METHODS ： This study was carried out in the Department of Neurology, Huashan Hospital Affiliated to Fudan University during June 2003 to August 2004. （1） Cell culture and experimental grouping： PC12 cells were cultured according to the method from Peng et al, then were randomized into 3 groups; blank control group, MPP^＋ group and insulin group. （2） Detection of relative survival rate of cells： The relative survival rate of cells at different MPP^＋ final concentrations （0, 50, 100, 200, 300, 1 000 μmol/L） and at different culture time （0, 4, 8, 12, 18, 24 hours） in the 300 Fmol/L MPP^＋ group and different concentrations of insulin （0, 15, 50, 100 nmol/L） in the insulin group was detected with MTT method according to the method from Hansen et al. （3） Observation of cell apoptosis： After stained by HOECHST 33258, the apoptotic cells were observed under the fluorescence miscroscope with the method from Chen et al. （4） Dection of apoptotic rate of cells： Apoptotic rate of cells was detected with flow cytometry according to the method from Zhang et al. （5） The expression of tyrosine hydroxylase （TH） mRNA in PC12 cells was detected with RT-PCR methods according to the modified method from Peng et al. MAIN OUTCOME MEASURES ： Comparison of relative survival rate, apoptosis rate, the expression of IR mRNA and TH mRNA and cell apoptosis. RESULTS： （1） After 12-hour incubation of 100, 200, 300 and 1 000 μmol/L MPP^＋, the relative survival rate of PC12 cells was （72.88±2.91）%, （60.64±0.81）%, （54.56±0.76）% and （16.89±2.83）%, respectively, which was significantly lower than that of blank control group （100%, P 〈 0.05）; After 12, 18 and 24-hour incubation, the relative survival rate of PC12 cells was （54.56±0.76）%, （42.43±0.16）% and （23.56±0.17）% respectively, which was significantly lower than that of blank control group （100%, P〈 0.05）; When 15, 50 and 100 nmol/L insulin was pre-added to cells, the relative survival rate was （70.10±0.16）%, （78.01 ±2.43）% and （83.55±1.43）%, respectively, which was significantly higher than MPP^＋ alone [（54.56±0.76）%, P 〈 0.05]. （2） Appototic bodies were rarely seen in the blank control group, but densely gathered in the MPP^＋ group and were significantly decreased in the insulin group. （3） Apoptosis rate of PC12 cells in the MPP^＋ group was significantly higher than that in the blank control group [（36.56±0.89）% vs. （2.34±0.23）%, P〈 0.05], and that in the 15, 50, 100 nmol/L insulin group [（30.01±0.04）%, （24.23±0.37）%, （20.01 ±1.01）%, respectivelyl was significantly lower than that in MPP^＋ group （P 〈 0.05）. （4） The TH mRNA expression in PC12 cells in MPP^＋ group was significantly lower than that in blank control group; The expression of TH mRNA in insulin group was gradually increased in an insulin dose-dependent manner. There were no significant changes in the expression of IR mRNA under different experimental conditions. CONCLUSION： Insulin can resist MPP^＋-induced apoptosis of PC12 cells, lessen the damage of PC12 cells, but does not change the gene expression of target cell insulin receptor.

Minocycline protects the apoptosis of PC12 cells induced by 1-methyl-4-phenylpyridinium

Objective： To explore the protective effect of minocycline on the apoptosis of cellular parkinsonism models induced by MPP^＋ . Methods： Using PC12 cells as the apoptotic model of dopaminergic neurons, MC and MPP^＋ were added into the culture medium of PC12 cells, and using MTr to assay the cell viability and metabolic state; The cells apoptosis was assayed by electrophoresis method and using flow cytometry FACS to assay the apoptosis ratio. Results： Added the MPP^＋ to get the concentration of 10μmol/L, the cellular parkinsonism model of apoptosis had been prepared. The pre-treatment of MC （ 100/μmol/L） could significantly increase the PC12 cell viability. The apoptosis ratio of MC＋MPP^＋ group was significantly lower than that of MPP^＋ group, but was still significantly higher than that of control group. Conclusion： MC may protect the cell apoptosis induced by MPP^＋ to some extent.

Protective effects of components of the Chinese herb grassleaf sweetflag rhizome on PC12 cells incubated with amyloid-beta42

The major ingredients of grassleaf sweetflag rhizome are β-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms of β-asarone and eugenol, components of the Chinese herb grassleaf sweetflag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations（between 1 × 10–10 M and 1 × 10–5 M） of β-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions（1 × 10–6 M β-asarone and eugenol）. The survival rates of PC12 cells significantly increased, while expression levels of the m RNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl m RNA increased. In addition, the combination of β-asarone with eugenol achieved better results than either component alone. Our experimental findings indicate that both β-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.

Eldepryl prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigral neuronal apoptosis in mice

Objective To study the apoptotic effects of 1 methyl 4 phenyl 1,2,3,6 tetrahydropyridine (MPTP) on the nigral dopaminergic neurons of mice and 1 methyl 4 phenylpyridium ion (MPP +) on pheochromocytoma (PC12) cells, as well as the antagonism of Eldepryl against MPTP's apoptotic effect Methods Three groups of C 57 BL mice were treated with MPTP, Eldepryl plus MPTP and normal saline, respectively, for 7 days before performing TUNEL (terminal deoxyneucleotidyl transferase mediated dUTP x nick end labeling) and FACS (fluorescence activated cell sorting) analyses of neuronal apoptosis in the substantia nigra The same tests were employed in cell culture to examine apoptosis in PC12 cells treated with MPP +, MPTP or PBS Results Intraperitoneal administration of MPTP 30?mg/kg could induce nigral apoptosis, and oral use of Eldepryl prior to MPTP treatment could completely prevent the nigral apoptosis caused by MPTP MPP +, an intermediate metabolite of MPTP, could lead to the apoptosis of PC12 cells, whereas MPTP itself had no such effect on PC12 cells Conclusions The experiment indicated that the neurotoxin, MPTP, might cause the death of nigral neurons through a mechanism of apoptosis and this effect might be mediated by its bioactive intermediate metabolite MPP + Eldepryl could protect the neurotoxicity from MPTP