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The Role of Autophagy Change in Manganese-induced Cytotoxicity of PC12 Cells

Author: YaoZuo
Tutor: LuoWenJing；CaiTongJian
School: Fourth Military Medical University
Course: Occupational and Environmental Health
Keywords: Manganese PC12 cells Autophagy
CLC: R114
Type: Master's thesis
Year: 2011
Downloads: 61
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Abstract

[Background] Manganese is abundant in the natural elements of the earth's crust, is an essential trace element. However, excessive manganese exposure is a recognized environmental health risk factors, can induce a variety of neurological symptoms similar to Parkinson's disease (Parkinson's disease, PD) extrapyramidal dysfunction especially. Environmental manganese pollution and the resulting health problems is an important public health problem. Eukaryotic cells degrade proteins and dysfunctional organelles important way from the bite - lysosomal pathway (autophagy-lysosome pathway ALP), with the ubiquitin - proteasome pathway (ubiquitin-proteasome pathway, UPP) together in the maintenance of intracellular play an important role in the process of homeostasis. In recent years, the study results show and the ALP function abnormalities play a key role in the occurrence of a variety of degenerative diseases of the nervous system. ALP function decline will lead to the abnormal accumulation of a variety of neurotoxic protein in neurons, and lead to further aggravation of neurodegeneration. Conversely, too high a level of autophagy can lead to degeneration and necrosis of neurons and plays a key role, especially in the neurotoxic substances induced toxicity process. [Objective] To establish manganism cell model neuron autophagy levels of manganese, and further study the autophagy level of change in the role of manganese-induced neurotoxicity process. The results are expected to provide ideas to further elucidate the molecular mechanisms of manganese neurotoxicity, as well as proposed new manganese neurotoxicity protective measures. [Methods] 1. Use of PC12 cells to establish manganese-induced DA neuronal damage in vitro model. Using flow cytometry, MTT, LDH activity detection method for determination of cell viability and cytotoxicity. Electron microscopy, immunofluorescence staining, Western Blot, etc. detection autophagy level change. 4.Western Blot detection autophagy substrate α-synuclein expression levels change. [Results] 1. The manganese poisoning vitro model MTT assay results show manganese role the same time, with the role of the higher concentration, cell viability decreased. The LDH activity detection results show that the degree of injury of PC12 cells gradually aggravated with the increased concentration of manganese role. Flow cytometry analysis showed that manganese after cell death and increased with increasing concentrations of manganese role. 2. PC12 cells induced by manganese-bite level change in the induced cell injury, manganese-induced change of the level of autophagy. Electron microscope showed that autophagy ultrastructural changes 300μmol / L manganese after 24h. MDC staining and EGFP-LC3 transfection typical autophagic morphological changes can be observed. Western Blot results show that the expression of microtubule-associated protein LC3-II/LC3-I autophagy-related gene Beclin 1 300μmol / L manganese role 12h, 24h showed an increasing tendency. Further study found that the level of manganese-induced autophagy change was mainly due to increased autophagosome generation and lysosomal degradation function abnormalities. Autophagic vesicles in PC12 cells exposed to manganese (Avs) can significantly reduce self-autophagy inhibitor 3-MA; lysosomal inhibitor Leu inhibits lysosomal degradation function of autophagy increased body quantity. 3 Since the role of macrophages in the manganese-induced injury in PC12 cells in preliminary study MTT, LDH and flow cytometry results, since the decline of the macrophage enhancer ease the manganese-induced PC12 cell viability, LDH release, and mortality increased autophagy inhibitors exacerbate manganese-induced cell damage; Conversely, inhibit autophagy enhancers manganese-induced cell damage. , Manganese can induce the expression of α-synuclein level increased and enhanced autophagy can inhibit its expression; Conversely, inhibition of autophagy promotes α-synuclein overexpression. Conclusion of PC12 cells induced proliferation inhibition, injury, and mortality, manganese can be induced by the increased level of autophagy. In the process of change in the level of manganese-induced autophagy, self-the macrophages generated increased rather than lysosomal degradation function decline has played a major role. 3. Increased level of manganese-induced autophagy is may be a compensatory protective effect, inhibition of autophagy may enhance its toxicity damage effect enhanced autophagy inhibited manganese-induced neuronal damage. The phenomenon is more pronounced. Promote autophagy, dyeing manganese group of α-synuclein expression levels decreased, while inhibition of autophagy level increased the expression of α-synuclein. Combined with the results of previous studies, we hypothesized that the enhancement and inhibition of autophagy manganese-induced toxicity in PC12 cells may be a certain correlation with the abnormal accumulation of α-synuclein degradation. In summary, this study is preliminary clarify manganese on dopaminergic neurons from macrophages level and its role in manganese-induced neurotoxicity results will further elucidate the molecular mechanism of manganese neurotoxicity and explore effective protective measures to provide new ideas and theoretical basis.

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CLC: > Medicine, health > Preventive Medicine,Health > Health - basic science > Health Toxicology
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