Protective Effects Of Green Tea Polyphenols On PC12 Cells From Rotenone Induced Damage

Objective : To investigate the possible protective effects and underlying mechanisms of GTPs(Green tea polyphenols), an natural antioxidant,on rotenone induced PC12 cells apoptosis.Methods:Rotenone was used on high differentiated PC12 cells to induce apoptosis.Four groups were analyzed:GTPs group(GTPs5, 15,45μg/ml + rotenone),selegiline group (selegiline +rotenone),control group(culture medium+ rotenone)and blank control group(culture medium only).1. Morphological changes of PC12 cells were visualized under reversal phase microscope.MTT assay was used to evaluate the PC12 cells viability. Lactate dehydrogenase (LDH) in the culture medium and in the cells was measured by spectroscopy respectively. Apoptotic cell ratio and cell cycle was detected with flow cytometer microscope.Hoechst33258 dyeing was used to detect cell apoptosis. Dopamine content was also examined.2. Malondialdelyde (MDA),superoxide dismutase (SOD) and reduced glutathione(GSH)in the cells were measured by spectroscopy respectively. Caspase-3 activity were examined by fluorospectrophotometer.Western blotting methods were used to examine express of caspase-3 . Bcl-2 and Bax mRNA expression were detected by RT-PCR.The results were analyzed with statistical methods.Results:1. Under reversal phase microscope,the normal cells differentiated obviously,grew well. Rotenone treated group cells appeared morphological changes,apoptosis and necrosis were also seen.GTPs at each concentration(5, 15,45μg/ml) treating PC12 cells increreased the cell viability compared with Rotenone group at the concentration of 2.5μmol/L in vitro(P<0.01).Cell viability of selegiline treatment group was higher than that of rotenone control group(P<0.01).Compared with the rotenone group , LDH leakage in GTPs and selegiline group were higher (P<0.01).Apoptotic ratios of GTPs at different concentration and selegiline group were significantly lowered compaered with rotenone group (P<0.01).Hoechst33258 dyeing displayed that GTPs and selegiline could protect PC12 cells from rotenone induced apoptosis. After GTPs treatment, the content of DA was increased in PC12 cells induced by rotenone (P<0.01).2. MDA levels increased significantly(P<0.01),while SOD activity and GSH levels decreased significantly (P<0.01) in rotenone induced group compared with those in the normal group.GTPs and selegine could reverse the effect,in which SOD activity and GSH increased significantly (P<0.01),while MDA levels decreased significantly (P<0.01) compared to rotenone. GTPs group reduced the caspase-3 activity compared with the rotenone model group . RT-PCR revealed that GTPs and selegiline increased the level of Bcl-2 mRNA,reduced the expression of Bax mRNA.Western blotting suggested that GTPs increased the expression of Caspase-3 protein.Conclusion:These results demonstrated that GTPs could protect PC12 cells against rotenone-induced damage and apoptosis . The neuroprotective effect might be related with the function that GTPs can reduce activity of apoptotic protein, reduce the oxidative damages and retain the DA dosage.