Proteomic Studies Of Model Of Parkinson Disease Induced By 6-Hydroxydopamine In PC12 Cells

Parkinson disease (PD) is a chronic neurodegenerative disorder of middle- aged and senile humans. It is characterized by the selective degeneration and deficiency of dopaminergic (DA) neurons, the emergence of eosinophilic inclusion (Lewy body) in survival dopaminergic neurons. The death mechanism of DA neurons is not fully known so far. For a long time, researchers have been attaching importance to the hypothesis that internal and external neurotoxins start the degeneration and death of DA neurons, and finally result in PD. As the neurotoxin similar to DA, the effect of 6-hydroxydopamine (6-OHDA) on distinctively damage the central DA neurons has been extensively recognized.This research establishes the international-standard PD model in which 6-OHDA induces the apoptosis and death of PC12 cells. It preliminarily probes into the toxic effect mechanism of 6-OHDA, so as to provide the reliable foundation for the original research on 6-OHDA's toxic effect mechanism on the proteomic levels. One of the essential problems of PD is to find its key proteins and mark proteins, then define the cause and precise mechanism of the disease, so that the early diagnosis and the high-flux screen of the remedial medicines can be realized. The methods and technologies of proteomic studies have brought new hope to this research area. On the base of traditional 2D electrophoresis technology, combining the multiple fluorescence analysis methods, differential gel electrophoresis (DIGE) separates several different fluorescence-lablled samples on the same gel and introduces the inter-gel standards for the first time. Thus, the accuracy, reliability and repeatability have been greatly improved. It has become one of the most reliable and accurate technologies in proteomic studies.To deeply explore the mechanism of PD, on the base of establishing the model of PD induced by 6-OHDA in PC12 cells, this research makes use of DIGE and MALDI-TOF mass spectrometry (MS) to select the related proteins, so as to disclose the mechanism of PD at the protein level and provide new valuable clues for the early diagnosis of PD and its remedial medicines.This research divides the PC12 cells from the same batch into the experimental groups and the control groups. Add 6-OHDA (stock solution in ascorbic acid). The final concentrations are: 25μM, 50μM, 100μM, 150μM and 200μM. The control groups are added the same amount of ascorbic acid. 6h, 12h, 24h and 48h later, apply MTT and Hoechst 33342 respectively to check the toxic effects of 6-OHDA on PC12 cells. Extract the total protein of the PC12 cells in the control groups and the PC12 cells treated by 100μM 6-OHDA for 24h, use DIGE to obtain the differential information from the protein points. In the end, employ MS technology to identify the differential proteins.Applying MTT, this research finds that 6-OHDA leads to the lower survival rates of PC12 cells. By Hoechst 33342, the typical apoptosis morphological changes of the cell nucleis can be found, indicating that 6-OHDA can induce apoptosis in PC12 cells. Meanwhile, within certain concentration ranges (25μM-200μM), there exists obvious time and dosage dependant. In proteomic studies, through MS analysis and database searching, 11 differential proteins and 15 differentail protein points are identified. The proteins indicating increase include glucose regulated protein 58KDa (GRP58), glucose-regulated protein 75 (GRP75), glucose-regulated protein78 (GRP78), glucose-regulated protein 94 (GRP94), heat shock 60KD protein (Hsp60), calcium-binding protein 1 (CaBP1), prolyl 4-hydroxylase beta polypeptide (P4H beta polypeptide), annexinⅤ, aldehyde reductase (AR) and alpha glucosidase II alpha subunit. The differential protein indicating decrease is heat shock 27 KD protein (Hsp27). Among the identified proteins, eight are molecular chaperones: GRP58, GRP75, GRP78, GRP94, Hsp60, CaBP1, P4H and Hsp27; Among them, six are heat shock proteins: GRP58, GRP75, GRP78, GRP94, Hsp60 and Hsp27. Except that Hsp27's indication has passed the peak and started to decrease, after the long-time interference of 6-OHDA, other molecular chaperones are all indicating increase, which has protective effects in endoplasmic reticulum stress (ERS). It can promote the functional restoration of endoplasmic reticulum (ER), stabilize the calium ion level, help to accurately fold, decorate and operate the accumulated proteins in ER. Moreover, it can be contributive to degrading the wrongly folded proteins so as to hold back the apoptosis of the cells. The dramatic increase in GRP78 and so on is the symbolized change of ERS, which intensely indicates the significant part of ER apoptosis in the 6-OHDA induced cell's death. Traditionally speaking, 6-OHDA mainly damages the mitochondrial respiratory chains, 6-OHDA induces apoptosis mainly through mitochondrial routes. This research has provided new evidence and ideas for the study of the toxic effects mechanism of 6-OHDA. What is more, two enzymes related to energy metabolism have also been recognized: AR and alpha glucosidase II alpha subunit. AR can reduce the fatty aldehyde, aromatic aldehyde and the toxic aldehydic compositions. It can catalyze aldehyde to degrade into suitable alcohol to display the protective effects against aldehyde and oxidative stress. The increase of AR may help to reduce the oxidation induced apoptosis. Alpha glucosidase II alpha subunit has the hydrolysis enzyme activity. It participates in the sugar metabolism of an organism, which plays an important role in maintaining the organism's normal physiology. The increase of alpha glucosidase II alpha subunit shows that there might be dysfunction glycosylation in PD models induced by 6-OHDA in PC12 cells. Bisides, this research has identified one protein related to apoptosis, annexinⅤ, which can inhibit the activity of phospholipase A2 and protein kinase C, take part in the activities of cystoskeleton, adjust the functions of membrane receptor, transmit mitotic signals, and promote cell secretion. In addition, annexinⅤcan form the high differential combination with phosphatidylserine (PS). When the apoptosis begins, PS will distribute over the surface of a cell membrane from the inner layer of the plasmolemma. The combining points between annexinⅤand cells increase, so annexinⅤis the sensitive indexes in the early period of apoptosis. At present, the fluorescence analysed annexinⅤhas been the significant method of identifying apoptosis. The excessive expression of the annexinⅤfurther proves that 6-OHDA has induced apoptosis in PC12 cells.For the first time, this research has analysed and identified the changes of the protein level in the PD model induced by 6-OHDA in PC12 cells. It has also explored differential expressing proteins'possible mechanism in the PD model induced by 6-OHDA in PC12 cells. Among the identified differential proteins, GRP94, CaBP1, AR, annexinⅤ, alpha glucosidase II alpha subunit, Hsp60, GRP58 and P4H beta polypeptide have not be found in the previous research on the PD model induced by 6-OHDA. The findings of this research have supplied new valuable clues to PD mechanism and the target selection for the PD remedial medicines. These proteins might become the new candidate targets of medicine treatment.