| Objective:To investigate the protective effects and its mechanisms of Cordycepin （Cor） on the apoptosis of PC 12 cells induced by rotenone （Rot）.Methods:After adding different doses of Rot into PC 12 cells, the optimal concentration of 1μmol/L was selected to establish the PD cell model in vitro. The experimental groups were divided into three groups after observing the effect of Cor itself on PC12 cells’ normal proliferation:（1） control group:0.01% DMSO; （2） model group: 1μmol/L Rot; （3） Cor group:0.4×10-6,2.0×10-6 and 8.0×10-6 μmol/L Cor were simultaneously administered with 1 μmol/L Rot, respectively. Cell viability was determined by MTT assay. LDH release was measured by colorimetric determination. Cell apoptosis was detected by DNA Ladder, the apoptotic morphology and the ultrastructure of cell were observed by Hoechst 33258 nuclear staining method and transmission electron microscopy（TEM）, and apoptosis rate was analysed by flow cytometry, to explore whether Cor play a protective role on Rot-induced PC 12 cell apoptosis. mRNA and protein expression of apoptosis related protein Bax, Bcl-2, Bcl-xL, caspase-3 were determined by real-time fluorescence quantitative PCR （RT-qPCR） method and western blotting method, to study the protection mechanism of Cor on PC 12 cell apoptosis.Results:（1） Preparation of PD cell model with Rot:After adding different concentrations of Rot （0.1,0.25,0.5,0.75 and 1 μmol/L） into PC12 cells, the OD values decreased significantly in different growth time （24 h,48 h,72 h）, which were extremely significant （F=367.232, P＜0.001; F=2068.715, P＜0.001）. Cell survival rate also decreased along with the time of culture and the increase of doses of Rot. Two factor analysis of variance showed significant interaction between the drug concentration and the time of culture （F=87.273, P＜0.001）. The results shown that the cell survival rate reduced to 52.9% at 72 h after 1 μmol/L Rot added into PC12 cells. Thus, the current dose of Rot （1 μmol/L） was chosen to establish the PD cell model in the subsequent experiments as follows.（2） The effect of Cor on the proliferation of normal PC12 cells:normal PC12 cells were incubated with a series of concentrations of Cor (10-6,10-5,10-4,10-3,10-2,10-1,1,101,102 μmol/L) for different time （24 h,48 h,72 h）, the OD values showed significant differences in the concentration level and time level （F=210.141, P＜0.001; F=549.775, P＜0.001）; two factor variance analysis also showed significant interaction between the concentration of Cor and the time of culture （F=26.553, P＜0.001）:Cor at lower concentrations (10-5～10-3 μmol/L) could promote the proliferation of PC12 cells at 24 h （P＜0.05） and could not do at some other time （P>0.05）; However, Cor at high concentrations （1～102 μmol/L） could inhibit the proliferation of PC12 cells significantly at different times （P＜0.05,0.01,0.001）. So, Cor at lower concentrations were used in the subsequent experiments.（3） The effects of Cor on cell viability and release rate of LDH in PD model:Compared with the control group, the cell viability decreased （P＜0.001） and the release rate of LDH increased significantly after 1 umol/L Rot treatment （P＜0.01）. After the model cell was incubated with Cor at 0.4×10-6,2.0×10-6 and 8.0×10-6 μmol/L for 24 h,48 h and 72 h, respectively, the cell viability increased and the release rate of LDH decreased, with significant differences at the time level （F=201.495, P＜0.001; F=78.952, P＜0.001）, so did those at the concentration level （F=68.296, P＜0.001）. F=65.281, P＜0.001), and there were also significant interaction between the concentration of Cor and the time of culture （F=8.237, P＜0.001; F=7.054, P＜0.001）. Compared with the model group, cell viability increased significantly and release rate of LDH decreased significantly in Cor group along with the increasing of Rot concentrations and the time of culture （P＜0.05）. The EC50 for improving cell viability among 10-8～10-4 μmol/L Cor at 24 h,48 h and 72 h were 5.6×10-6,4.5×10-6 and 4.8×10-6μmol/L, respectively. The results showed that Cor had a protective effect on PC 12 cell injury induced by Rot.（4） DNA ladder detection:The typical DNA ladder bands were found when cells treated with Rot for 48 h. Its size was about 180-200 bp and integer times. But these typical features did not be found in cells of control group and drug groups with large fragment genomic DNA bands. It was showed that DNA fragmentation reduced after Cor’s intervention for 48 h.（5） Hoechst 33258 nucleus staining:Morphologically, a normal structure was observed in cells of control group. The nuclear chromatin was homogeneous and intact, and the nuclei were blue. In contrast, Rot-treated cells exhibited apoptosis in the model group, including nuclear chromatin condensation, aggregation, nuclear fragmentation, and the dense blue bright dots in nucleus. The above morphological changes could be improved by Cor at concentration of 0.4×10-6,2.0×10-6 and 8.0×10-6 μmol/L at different time. Compared with the model group, the cells with bright blue nuclei decreased in the drug groups, which was the most obvious in Cor group of 8.0×106 μmol/L.（6） The changes of cell ultrastructure:TEM results showed a normal cell morphology in the control group, including abundant microvilli on the surface, a clear nucleus structure, and the abundant euchromatin and organelles in cytoplasm. In contrast, Rot-treated cells exhibited apoptosis in the model group, characterized by cell membrane shrinking, chromatin condensation and margination, and hyperchromatic apoptotic bodies. The ultrastructure of PC 12 cells tended to be normal after Cor intervention manifested by the clear cell membrane and nuclear chromatin, intact organelles in cytoplasm, and a few microvilli on the surface. The most obvious effect was observed in 8.0×10-6 umol/L Cor group, with the normal cell structure and abundant microvilli.（7） Apoptosis rate determination:The results determined by flow cytometry indicated that in the model group, the early and late apoptotic cells increased significantly （P＜0.001） and total apoptosis rate also increased significantly （P＜0.001）, compared with the control group. Late apoptotic cells and total apoptosis rate decreased significantly after Cor treatment. The inhibition effect enhanced with the increase of Cor dosage, and there was significant difference in total apoptosis rate among the three levels of concentration （P＜0.001）.（8） The mRNA expression of Bcl-2, Bcl-xL, Bax and Caspase-3:Compared with the control group, the mRNA expression of Bax in the cells of model group significantly increased and the mRNA expression of Bcl-2 and Bcl-xL significantly decreased （P＜0.001）. Compared with the model group, Bax mRNA expression decreased significantly in the higher dose (2.0×10-6 and 8.0x 10-6 μmol/L) groups （P＜0.001）. after the intervention of Cor. The mRNA expression of Bcl-2 and Bcl-xL significantly increased （P＜0.05, P<0.01）, especially at the dose of 8.0×10-6 μmol/L Cor. The above results indicated that the Cor had significant effects of inhibition or promotion on the mRNA expression of Bax, Bcl-xL and Bcl-2 in PC 12 cells in a dose-dependent manner. Compared with the model group, Caspase-3 mRNA expression had no significant change （P>0.05） in Cor group, suggesting that Caspase-3 did not change significantly at the level of transcription.（9） The protein expression of Bcl-2, Bcl-xL, Bax and Caspase-3 determination: Ccompared with the control group, protein expression of Bax increased significantly in the model group （P＜0.01）, and the protein expression of Bcl-2 and Bcl-xL decreased significantly （P＜0.01, P＜0.01） in the model group. The effects were reversed after the addition of Cor in a dose-dependent manner. The Bax/Bcl-2 ratio also increased in a dose-dependent manner. The protein expression of cleaved caspase-3 in model group increased significantly compared with the control group （P＜0.001）, while the effect reversed after Cor treatment （P＜0.001）, especially at the dose of 8.0x 10-6 μmol/L. The results suggested that the protective effect of Cor on PC12 cell apoptosis may be related to altering protein expression of Bax, Bcl-2, Bcl-xL and inhibiting Caspase-3 activity.Conclusion:Cor had a potential protective effect on apoptosis induced by Rot in PC12 cell. The protective mechanism would be related to the inhibition of expression of Bax, the promotion of expression of Bcl-xL and Bcl-2, and the inhibition of Caspase-3 activity. |
