Establishment Of A Parkinson’s Disease Modol In Chronic MPP~+-Injured SH-SY5Y Cell Line And The Protective Effect Of Polypeptide From Velvet Antler Of Cervus Nippon

BackgroundParkinson’s disease (PD) is a common neurodegenerative disease in agedpopulation. The growing incidence of PD is a socio-economic problem affecting lifequality of PD patients. The syptoms of PD inclue tremor, bradykinesia, rigidity andflexed posture. Pathological features of PD implicated loss of dopaminergic neuronsin substanitial nigra, which cause the decreased dopamine level in the striata. Untilnow, the mechanism underlying PD onset is not fully understood. Most of the in vivoand ex vivo studies had illustrated that some of the mechanism involved in the PDpathogenesis, including mitochondial dysfunction, oxidative stress, ubiquitin-proteasomal pathway, etc. Recently, a misfolded protein related endoplasmicreticulum stress (ER stress) had been most intensively studied for its actions toneuronal cell death. Thus, understanding the process of ER stress is of great helpful toexplore novel neuroprotective agent for PD treatment. Clarifing the mechanisms ofPD onset and development of neuroprotective therapeutics are largely depend onestablishment of disease model, especially model in vitro. It is known that PD is achronic and progressive disease, but acute lesioned model in vitro was most commonused in domestic studies for PD. Our study indicated that the acute lesioned modelfailed to relect the chronic and progessive process of PD onset. Hence, a chroniclesioned model in vitro should be attentioned considerably in order to reveal the actualmechanism of PD and develop novel neuroprotective agents. Based on theseunderstandings,1-methyl-4-phenylpyridinium (MPP~+), a neurotoxin most commonlyused in establishment of PD model in vitro and human neuroblastoma SH-SY5Y cellline had been utilized to establish PD model. In this study, SH-SY5Y cell underoptimized acute or chronic MPP~+exposure was selected in comparison to features ofacute and chronic lesioned model. Based on these data, the chronic MPP~+lesionedmodel was selected to study ER stress mediated cell death, and the neuroprotectiveeffect of velvet antler polypeptides (VAPs), the peptides extracted from cervus nippon,was evaluated to develop the potential therapeutic agent for PD treatment. Methods1. VAPs was obtained from fresh velvet antler using colloid mill, ammonium sulfateprecipitation, lyophilizing procedure. For further study, SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), laser desorption ionization time-of-flight massspectrometry (MALDI-TOF-MS) and amino acid composition analysis wereperfomed to characterize the physical and chemical features of VAPs.2. For acute MPP~+lesioned model in vitro, MPP~+with different concentrations wereexposed to human neuroblastoma SH-SY5Y cell line for24h,48h and72hrespectively. The half maximal inhibitory concentration (IC50) of MPP~+in SH-SY5Yand protective effect of VAPs against MPP~+cytotoxicity were measured using MTTassay. For acute MPP~+exposured model, a72h exposed model was selected. Forfurther study in this model, cells were divided into control, model and VAPs treatedgroup. Control cells were incubated with midium containing10%fetal bovine serum,MPP~+-treated cells were incubated with120.9μM MPP~+, VAPs group cells wereincubated with62.5、125、250μg/mL VAPs in the presence of120.9μM MPP~+. Cellviability was detected with MTT assay; Cell apoptosis rate and mitochondrialmembrane potential (ΔΨm) were measured using Hoechst33342and rhodamine123staining respectively; Intracellular reactive oxygen species (ROS) level was assayedwith H2DCFDA staining; Western blot and immunocytochemistry were performed todetect the level of ER stress related proteins, such as caspase-12, heavy-chain bindingprotein/glucose regulated protein78(Bip/GRP78), C/EBP homologous protein,CCAAT/enhancer binding protein homologous protein (CHOP) and c-Jun N-terminalkinase/Stress-activated protein kinases (JNK/SAPK).3. For establishment of chronic MPP~+intoxication model, SH-SY5Y cells wereexposed to MPP~+for5days,10days and15days. Cell viability was measured withMTT assay; Cell apoptosis rate and ΔΨm were detected with Hoechst33342andrhodamine123staining; Intracellular ROS production was determined withH2DCFDA staining; Western blot and immunocytochemistry were processed forcaspase-12, GRP78, CHOP, phosphorylated JNK（p-JNK). Results1. Velvet antler polypeptides (VAPs)A water soluble fluffy white powder containing54%protein had been obtained. Thelaser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)analysis indicated that molecular weigh of most of the peptides in VAPs ranged from10kDa~20kDa. Further purification and MALDI-TOF-MS analysis characterizedpeptides with molecular weigh of3~11kDa, which enrihed in asparagine, serine,glutamic acid, glycine, alanine, valine, leucine, phenylalanine, lysine, arginine,proline, but no cystine, tyrosine, tryptophan detected.2. PD model received acute MPP~+exposure⑴120.9μM MPP~+decreased the cell viability of SH-SY5Y cell and increased cellapoptotic rate.125and250μg/mL VAPs protected against MPP~+-induced cell deathin a significant way, which was much apparent for72h protection. MPP~+lead to△Ψ mdecrease and ROS production, but VAPs inhibited these changes in SH-SY5Ycell induced by MPP~+.⑵MPP~+exposure result in a robust expression of caspase-12, and VAPs treatmentinhibited this process. GRP78, CHOP, p-JNK expression were not observed in cellsexposed to MPP~+. Collectively, these results show that a acute MPP~+exposurecharacterized with mitochondrial dysfunction and oxidative sress mediated cell death.VAPs counteract cell death induced by MPP~+. MPP~+incuced caspase-12expression ina significant way, but other RE stress associated proteins such as GRP78, CHOP andp-JNK were not apparent. It is conceivable that an acute MPP~+lesioned model in vitrocaused cell death depending on activation of mitochondrial pathway related caspasecascade and oxidative stress, but not determined by ER stress associated cell deathsignaling.3. Chronic MPP~+intoxication model⑴IC50values for5days,10days and15days MPP~+exposure were31.1μM,22.1μM and10.4μM respectively, which were used to establish MPP~+intoxication modelfor chronic exposure. MTT assay indicated500μg/mL VAPs protected against MPP~+induced decrease of cell viability.⑵In MPP~+intoxication model for chronic exposure, either5days exposure or10days,15days exposure influenced cell apoptosis rate or△Ψm. But ROS productionwas visualized using fluorescent probe in cells exposed to31.1μM MPP~+for5days. 22.1μM MPP~+or10.4μM MPP~+for10days and15days exposure had no effect onROS production. VAPs treatment inhibited the ROS level in MPP~+lesioned model for5days.⑶Western blot and immunocytochemistry indicated there was caspase-12, GRP78,CHOP and p-JNK expression in cells treated with MPP~+for5days. VAPs treatmentdownregulated these protein expression. But neither22.1μM MPP~+,10.4μM MPP~+exposure nor VAPs treatment for10days and15days induced ER stress associatedprotein expression, which indicated the chronic MPP~+exposure lead to ER stressactivation, but not initiation of mitochondrial pathway directly. These data show thatSH-SY5Y cell under31.1μM MPP~+exposure for5days and120.9μM exposure for72h appeared complate different cell signaling phenotypes. Our data identified acuteMPP~+injury induced mitochondrial dysfunction and oxidative stress mediated celldeath, which had been substantiated by cell apoptosis bodies in response to MPP~+exposure. A chronic MPP~+exposure induced ER stress and oxidative stress, but failedto result in cell death. We recognized that a chronic MPP~+intoxication model canmimic the progressive and long term neurodegenerative process of PD onset muchbetter than a acute and short term model. In conclusion, our model in vitro is ideal forstudying ER stress associated pathways. VAPs protect against chronic MPP~+intoxication and the mechanism underlying supression of ER stress associatedproteins activation.