| The Parkinson,s disease is a kind of commonly central neurodegenerative diseases. There is still no effective drug to cure it radically. The injury model of Parkinson's disease in vitro was made by 1-methyl-4-phenylpyridinium ion (MPP+) in PC12 cells. Ginsenoside monocase with protection of dopaminergic neurons against injury and apoptosis was screened from 9 ginsenosides, providing a feasible experimental basis in vitro for further animal research and and hence possible drugs candidate in the development of new preventive and therapeutic drugs for PD.1,Dose and time-effect relationship of inhibiting effects of MPP+on the viability of PC12 cell : The cell viability was determined by MTT assay. Optical density of cell decreased gradually after MPP+ was added into PC12 cells for 12h,24h or 48h. Compared with the model group, optical density of cell was significantly reduced only in the high dose group at 12h (P<0.05),while optical density of cell showed much significant difference at 24h and 48h(P<0.001). Cell viabilities were the lowest at 48h. It showed dose–dependent decrease when PC12 cell was treated with different concentrations of MPP+(100, 200,300,400,500μmol/L). Cell viabilities was 98.8%,93.4%,91.7%,90.7%,86.6% respectively after 12h; it was 91.6%,87.4%,84.8%,78.2%,73.7% after 24h and 91.9%,84.4%,70.9%,56.4%,50.2% after 48h. The results indicated that MPP+ could inhibit cell viabilities and induce injury of PC12 cell, which was dose- and time- dependent.2. The effects of 9 ginsenosides on the injury of PC12 cell induced by MPP+: Optical density of PC12 cell was detected after treating with different concentrations of 9 ginsenoxides(0.1562,0.3125,0.625,1.25,2.5,5.0,10,20μmol/L)and 400μmol/L MPP+ for 48h.. In model groups with 400μmol/L MPP+ cell viabilities was decreased to 62.0%±1.9%(P<0.001). Compared with model group, ginsenosides Rg3,Rb3,Rg2 and Re enhanced cell viabilities remarkably. Emax ( the highest cell viabilities )was 95.9%,92.0%,76.5%,76.1%, and EC50 was 0.806,0.309,0.827,1.701μmol/L respectively. Among the rest five ginsenoixedes : the cell viabilities were increased slightly by Rb2 only with low concentrations (0.3125,0.625μmol/L)(P<0.05). At low concentration(0.3125,0.625μmol/L), Rc still showed inhibition by contrast with slight promotion of cellular viability at 20μmol/L. Rd,Rh1,Rh2 could make cell viability decline distinctly(P<0.05 , 0.01) when the concentration range was in 0.1562μmol/L～2.5μmol/L. Compared with ginsenosides Rg3,Rb3,Rg2 and Re, these five ginsenoixedes had less influence on cell viabilities. It was suggested that ginsenosides Rg3,Rb3,Rg2 and Re could alleviate markedly the cell damage caused by MPP+.3. The effects of ginsenosides Rg3 and Rb3 on apoptosis of PC12 cell induced by MPP+: The cell apoptosis was detected by Annexin V-FITC/PI. Morphological change of apoptosis was examined by AO/EB staining. The ratios of apoptotic cells were increased from 39.0%±4.5% in control group to 51.2%±2.1% ( P<0.05 ) and 64.9%±2.7% (P<0.05) respectively, after treating with 300 and 400μmol/L MPP+ for 48h. MPP+ could not result in an remarkable increase in the ratio of early apoptosis PC12 cell , however, late apoptosis/death cells was increased markedly. At 24h the ratio of apoptotic cells was increased from 3.7%±1.2% in control group to 13.1%±1.7% in the group with 400μmol/L MPP+(P<0.05). It showed that MPP+ may induce apoptosis of PC12 cell, which was dose and time dependent.PC12 cell was treated with 400μmol/L MPP+ after treating with ginsenosides Rb3 or Rg3 for 24 and 48h. Then the cell apoptosis was detected and morphological change of apoptosis was observed .The cell apoptosis was a downtrend after treating with ginsenoside Rb3(2.5,5.0μmol/L) for 24h, but displayed no significant difference. At 48h the ratios of apoptotic cells were decreased from 64.9%±2.7% in model group to 59.8%±2.9%(P>0.05)and 37.3%±1.1%(P<0.01) respectively. It was decreased from 13.1%±1.7% in model group to 7.3%±2.0%(P>0.05)and 5.6%±1.7%(P<0.05)respectively after treating with ginsenosides Rg3(2.5å'Œ5.0μmol/L) for 24h. And it was decreased from 64.9%±2.7% in model group to 58.2%±1.6%(P>0.05)and 31.6%±3.2%(P<0.01)respectively at 48h. The results indicated that ginsenosides Rb3 and Rg3 may inhibit apoptosis in PC12 cell induced by MPP+, and there was a dose-effect relationship.4. The relationship between ginsenosides Rb3 and Rg3 induced inhibition of PC12 cell apoptosis and ROS: ROS in the cell was detected by flow cytometric analysis (FCM). The amount of ROS in PC12 cell was increased greatly after treating with 200 and 400μmmol/L MPP+ for 24h and 48h, assumes ROS ratio dependence and the time dependence relations. The ROS ratios were decreased from 1.742±0.07 in model group to 1.156±0.03 (P<0.05) and 1.182±0.08 (P<0.05) respectively after treating with ginsenosides Rb3(2.5 ,5.0μmol/L) for 48h. Ginsenosides Rg3 (2.5,5.0μmmol/L) also could restrain the ratio of the rising of the ROS, but had no significant difference when compared with model group. It showed that ginsenoside Rb3 could restrain the ratio of the rising of the ROS caused by MPP+, but had no dose-effect relationship.Rg3 could not effectively antagonize the rising of the ROS in PC12 cell caused by MPP+. It was suggested that ginsenosides Rb3 and Rg3 induced inhibition of PC12 cell apoptosis was nearly independent of ROS. |
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