Protective Effect Of Rapamycin On Rotenone-induced PC12 Cell Injuries And Its Relative Machanisms

Parkinson's disease (PD), also known as paralysis agitans, is the second largest neurodegenerative diseases. Its pathogenesis is unclear up to now. The inhibition of mitochondrial complex I, energy supply dysfunction, oxidative stress, dopamine metabolic disorder and dysfunction of protein degradation were known as the major mechanisms. Extensive studies demonstrated that the dopamine receptor agonist, monoamine oxidase inhibitor, excitatory amino acids inhibitor, neuropeptide, antioxidant, inflammatory reaction inhibitor, neurotrophic factors,immunosuppressive agents, estrogen and so on were protective to PD. Though some progresses were achieved, there is no completely effective medicine of prevention and cure of PD. Therefore, to explore new and effectively neuroprotective medicine is the focus and difficult point to prevent and cure PD.Rapamycin known as Sirolimus, belongs to macrolides compounds. Rapamycin is a kind of immunosuppressive agents, and its chemcial constitution is similar to FK506. Rapamycin combined with FKBP-12 forms the Rapamycin - FKBP-12- compound, which impacts on the mammalian target of rapamycin to inhibit the activity of mTOR to active autophagy to enhance degradation of protein aggregates resulting neuron protection. So, sirolimus is regarded as a potential therapeutic medicine in neurodegeneration.Based on the research progress and the results of the effects of rotenone leading to PD obtained in our lab, PC12 cells exposure to rotenone was established as PD model in vitro to investigate the protective effects and mechanism of rapamycin on PC12 cells treated by rotenone.OBJECTIVE:To investigate the neuroprotective effects of rapamycin on PC12 cells administrated with rotenone and to explore the possibilities of rapamycin asa medicine to prevention and cure of PD and its related mechanisms.METHODS:PC12 cells were cultured in vitro and treated with rotenone (50 nmol.L-1 for 24 h ) or rapamycin (25,50,100,200μg.L-1 for 12 h) + rotenone (50 nmol.L-1 for 24 h ). Morphologic changes of the cells were tested by the immunofluorescence method with 4',6-diamidino-2-phenylindole fluorescein staining. The apoptotic rates and the mitochondrial membrane potential (m) of the cells were determined by the method of AnnexinⅤ-FITC/PI and rhodamine123, respectively. Acridine orange fluorescein stain was used to observe the formation of the autophagosome. The protein expression of alpha-synuclein was detected with with cytoimmunity and immunohistochemisty. The formation of protein aggregates was examined with thioflavin S. The protein expression of Caspase-3, heat shock protein 70(HSP70) and microtubule-associated protein light chain 3(LC3) were detected by Western blot. The activity of SOD,GSH-Px and NOS were detected with biochemistry.RESULTS:1. An experimental PC12 cell exposure model to lower dose of rotenone has been successfully established in this subject. After treatment with rotenone at the concentration of 50 nano mole (nM) for 24h, three types of morphological changes in the damaged cells were observed in the cells nuclei: chromatin agglutination, nuclear condensation and nucleosome fegments. Compared with the control group, the apoptotic rate of the rotenone treated cells was significantly increased by 6.25% (P<0.01). The activities of SOD and GSH-Px, however, were obviously decreased (P<0.01).α-Syn concentration andα-syn aggregation in the PC12 cells treated with 50 nM rotenone were determined at 72h. Evident protein aggregation was appeared at 144h.2. Rapamycin treatment significantly improved the chromatin density and apoptosis rate of the cultured PC12 cells treated by rotenone, in a concentration-dependent manner. The apoptosis rate of PC12 cells decreased 40.2%(P<0.01) in the rotenone treatment groups compared with 200μg.L-1 rapamycin treatment groups.3. Rapamycin (25-200μg.L-1) treatment obviously improved the mitochondrial membrane potential compared with the rotenone treated cells. Rapamycin (200μg.L-1) obviously increased the mitochondria membrane potential for 317% (P<0.01)4. Rapamycin treatment inhibited the formation of protein aggregation, especially of the key protein of alpha-synuclein.5. Rapamycin treatment improved the protein expression HSP70, which was inhibited by rotenone. 3-methyladenine(3-MA) had antagonism effects induced by rapamycin on PC12 cells treated by rotenone. 6. The numbers of autophagysome increased in the PC12 cells exposed to rotenone with Rapamycin treatment , with a shifting of LC3ⅠtoⅡ. (P<0.01). 3-MA had antagonism effects induced by rapamycin on PC12 cells treated by rotenone.7. Activation of Caspase-3 protein was inhibited in the PC12 cells exposure to rotenone with rapamycin treatment , active fragment of p20K decreasing (16.2%(P<0.01)). 3-MA had antagonism effects by rapamycin on PC12 cells treated by rotenone.8. SOD and GSH-Px activity were significantly increased by rapamycin(50～200μg?L-1) in the PC12 cells exposed to rotenone, and the activity of NOS was markedly decreased compared with PC12 cells treated by rotenone only.CONCLUSION1. An experimental PC12 cell exposure model to rotenone has been successfully established in this subject. At the range of 50～100 nM, rotenone induced the fundamental toxic changes such as the shrunken nuclei, border irregularity, chromatin agglutination and nuclear condensation accompanied by appearance of nucleosome fragments and the protein aggragation. At the same time , the apoptotic rate of the cells increased and the activities of antioxidative enzenmes decreased.2. Cyto-chromatin enrichment, karyopyknosis and nucleosome fragment were inhibited in the PC12 cells exposed to rotenone with rapamycin treatment. Apoptosis rate decreased and mitochondrial membrane potentials increased, in a concentration-dependent manner. It was suggested that apoptosis in the PC12 cells induced by rotenone was inhibited partly by rapamycin treatment.3. The formation of autophagysome was promoted in the PC12 cells exposure to rotenone with rapamycin treatment, with a shifting of LC3ⅠtoⅡand with autophagy enhancement.of alpha-synuclein aggregates and abnormal mitochondrials. The release or activation of mitochondrial-related proapoptosis factor was reduced, and the cleaved Caspase-3 fragment descreased, which was suggested that Caspase-dependent apoptosis pathway was inhibited partly in the PC12 cells treated by rapamycin. 3-methyladenine could significantly abolish the shifting of LC3ⅠtoⅡand Caspase-3 activation in the PC12 cells treated by joint application of 3-methyladenine and rapamycin. These confirmed the protective effect of rapamycin was mainly through autophagy enhancement.4. The expression of HSP70 were obviously improved by rapamycin treatment in rotenone-induced cell, which indicated an upregulation of misfold protein refolding with a result of reduced ubiquitin-proteinase system stress.5. Rapamycin treatment could significantly reverse the effect of rotenone-induced depressed activity of SOD, GSH-Px and an increased activity of NOS. These could promote ROS clearance, RNS decrease in turn and lower the destruction of cellular membrane, particular mitochondrial membrane, as well as respiratory electron transport chain complex damage. It could also inhibit the direct damage of DNA by NO/ONOO-, avoiding exhaustion of NAD+, ATP during DNA recovery. This might be another protection of rapamycin on rotenone-induced neuron injuries.In conclusion, these findings indicates that there is two pathways in the protective role of rapamycin in rotenone-induced PC12 cell apoptosis. In one way, rapamycin could enhance degradation of protein aggregation together with innormal mitochondria. Furthermore, it reduces the releasing or activation of mitochondrial-related proapoptosis factors, which suggestes that autophagy-dependent pathway is upregulated and Caspase-dependent apoptosis pathway is inhibited partially. In another, rapamycin could clear intracellular ROS and reduce the RNS level effectively in rotenone-treated PC12 cell. Rapamycin could also increase the endogenous anti-oxidative enzyme activities and decrease NOS activity, which could result in a decrease the direct damage of NO/ONOO- to dopaminergic neurons.