| Part 1 6- hydroxydopamine(6-OHDA)-induced PC12 cell apoptosis andits possible mechanismObjective To explore the specific role of GSK3β in the 6-OHDA-induced apoptosis pathway and the possible mechanism of 6-OHDA inducing PC12 cell apoptosis using an in-vitro model of Parkinson’s disease established by PC12 cell damages induced by 6-OHDA.Methods A in-vitro model of Parkinson’s disease was established by PC12 cell damages induced by 6-OHDA. The viability and survival rate of PC12 cells following treatment with different concentration of 6-OHDA was determined by MTT assay and trypan blue dye exclusion test. Western blotting was used to detect: 1. total GSK3β and p-GSK3β levels; 2. Bax and β-actin levels(β-actin being the internal reference of intracytoplasmic protein); and 3. Bax and prohibitin levels(prohibitin being the internal reference of intramitochondrial protein).Results Treatment with 6-OHDA for 24 h provided dose-dependent(10, 20, 50,100 and 200μm) inhibition of the viability and survival rate of PC12 cells. Viability and survival rate of PC12 cells was significantly inhibited at the 6-OHDA concentration100μM.Western blotting showed that treatment with 6-OHDA reduced GSK3βphosphorylation in a time-dependent manner. When examining the presence of Bax in mitochondria by extracting mitochondria and cytoplasm, it was shown that treatment with6-OHDA reduced Bax in the cytoplasm while increased Bax in the mitochondria,indicating mitochondrial translocation of Bax induced by 6-OHDA.Conclusions 6-OHDA significantly reduced PC12 cell viability and promotedPC12 cell apoptosis. GSK3β plays a certain role in 6-OHDA-induced apoptosis. Treatment with 6-OHDA resulted in time-dependent reduction of GSK3β phosphorylation, and induced Bax translocation from cytoplasm to mitochondria, causing mitochondrial dysfunction which further led to cell apoptosis.Part 2: Protective Effects of Erythropoietin(EPO) against6-OHDA-induced PC12 Cell Apoptosis and its MechanismObjective To explore the mechanism of EPO inhibiting 6-OHDA-induced apoptosis by investigating the effects of EPO on GSK3β phosphorylation, mitochondrial translocation of Bax, Bcl-2 expression level and Caspase 3 activity in 6-OHDA-treated PC12 cells.Methods PC12 cells were pre-treated with TDZD-8(25μM) or different concentrations of EPO(0-10U/m L) before incubation with 100μM 6-OHDA.Cell viability following different treatments was determined by MTT assay and trypan blue exclusion test; Western blotting was used to detect the GSK3β phosphorylation level,total GSK3β level, intracellular distribution of Bax, prohibition level, and Bcl-2 andβ-actin levels; the proportion of apoptotic PC12 cells was estimated by Hoechst 33258staining; and Caspase 3 activity was determined using Caspase 3 colorimetric assay kit.Results EPO reduced 6-OHDA-induced growth inhibition in a dose-dependent manner(over the concentrations ranging from 1 to 10U/m L), and the GSK3β inhibitor4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione(TDZD8) markedly reversed6-OHDA-induced growth inhibition.EPO and TDZD-8 markedly reversed inhibition of GSK3β phosphorylation in the presence of 6-OHDA, and markedly suppressed 6-OHDA-induced mitochondrial translocation of Bax.6-OHDA did not alter the Bcl-2 expression level in cytoplasm, nor did EPO or TDZD-8 exhibit any effect on Bcl-2 expression level in cytoplasm. In contrast, 6-OHDAsignificantly suppressed Bcl-2 expression in mitochondira, which however could be reversed by addition of EPO and TDZD-8.EPO and TDZD-8 inhibited 6-OHDA-induced cell apoptosis. 6-OHDA markedly enhanced Caspase 3 activity, while EPO and TDZD markedly inhibited the enhanced Caspase 3 activity.Conclusions EPO inhibits 6-OHDA-induced apoptosis by promoting GSK3βphosphorylation in 6-OHDA-treated PC12 cells, reducing mitochondrial translocation of Bax, reserving suppressed Bcl-2 level in mitochondria, and suppressing Caspase 3 activity,which contribute to its neuroprotective effect. These indicate EPO’s good potential to be a neuroprotective agent. |
PDF Full Text Request