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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 Academyof 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, 24hours) in the 300 μmol/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 μ mol/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 )%, respectively] 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.CONCLUSTON: 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.