Experimental Study On MPP～+-treated PC12 Cells In Vitro: Arrested Proliferation And Possible Mechanism

Among adults, Parkinson's disease (PD) is the most common neurodegenerative movement disorder and second most common neurodegenerative brain disease which is characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. Substances that are toxic to dopaminergic neurons have been proposed as contributors to this neurological disease. l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) is among these dopaminergic neurotoxins. Studies showed that MPTP induces Parkinson's-like neuropathology in animals. In order to be active, MPTP must be converted into 1-methyl-4-phenylpyridinium ion (MPP+), the true toxic agent, by the monoamine oxidase B of the inner mitochondrial membrane.In recent years, it has been suggested that apoptosis is involved not only in physiological cell death during normal development but also in neurodegenerative diseases. There is evidence of apoptotic neuronal death in PD. MPP+ was reported to induce neural cell death with characteristics of apoptosis in various neuronal cells.Mitogen-activated protein kinases (MAPKs) are a family of proteinkinases playing a central role in signal transduction and thought to mediate diverse processes ranging from transcription of protooncogenes to programmed cell death (PCD). MAPKs include several serine-threonine kinases in three interrelated signal transduction cascades, and can be activated by stimuli such as growth factors, stress and inflammation. ERK pathway engages in regulating the proliferations and differentiations of cells. JNK and p38 cascades were usually activated under stress circumstances and correlate to several physiological and pathological courses. The parallel MAPKs cascades regulate and associate with each other.On the basis of former experiments, we have employed a model in vitro using the PC 12 cell line to study the toxicity of MPP+ to dopaminergic cells. We also explored the characteristics of MAPKs and relations between ERK and p38 cascade under the same conditions aiming to reveal the molecular mechanisms of neuron degeneration in the substantia nigra caused by MPTP or other PD related environmental exposure factors. Accordingly, research efforts have been directed toward understanding the etiology and pathogenesis of PD and other neurodegenerative diseases in the hope of developing a more effective preventative measures and therapy that will halt the neurodegenerative diseases and providing an appropriate referenced criterion of environmental exposure factors. MethodsPC 12 cells were co-cultured with MPP+ within a dose range of 100-700 umol/L. The inhibition of the cells during 7 days was determined by MTT assay and cell counting, and the cell growth curve was made. In the following experiments, MPP+ was administered during 4 days to the cells to take effects in a non-acute way. Then the morphological changes were observed by transmission electron microscope (TEM) and the apoptotic rate of the cells was monitored by flow cytometry analysis (FCM) at the same time. TUNEL-stain was also performed to detect apoptotic cells. Western-blot was used to test phosphorylation of ERK1/2 and p38 proteinsof MPP+-treated PC 12 cell in different time courses and doses. In addition, we also use PD98059 and SB203580 to block ERK and p38 cascade, respectively, and then observed the activation of MAPKs and apoptotic cells. ResultsMPP+ at different concentrations (100, 300, 500, 700 umol/L) could inhabit the growth and proliferation of PC 12 cells in a dose- and time-dependent manner and the cell growth curve was changed accordingly (P<0.01). After 4 days' exposure of MPP+, from 100-700 umol/L, the viability of cells decreased from 0.78 to 0.34 in comparison with the control. When co-cultured with 300 umol/L MPP+, some apoptotic changes of the cells were observed under transmission electron microscope. After the treatment of 100, 300 umol/L MPP+, FCM showed that the apoptotic rate of PC 12 cells was increased in a dose-dependent manner (P<0.01), and...