Protective Effect And Its Relative Mechanisms Of Quercetin By Inhibiting Oxidative Stress In Rotenone-induced U87 Cell Injuries

Parkinson’s disease(PD) is a common neurodegenerative disease that afflict the elderly patients. Oxidative stress is one of the important factors of the pathogenesis of Parkinson’s disease. Quercetin has a wide range of biological activities, including antioxidant, anti-tumor, anti-inflammatory and anti-bacterial, improve cardiovascular status. Whether quercetin has a neuroprotective effect on PD remains unknown.Therefore, with the modern technology cytology, this study aimed to explore the role of cellular oxidative stress and apoptosis on the protective effect of quercetin and its possible underlying mechanism. This study aims to use modern cytology technology,detection the protection of quercetin on cell oxidative stress and apoptosis induced by rotenone, in order to understand the quercetin’s targets and its mechanism from the microscopic view thus provide theoretical basis for the prevention of Parkinson’s disease.Trypan blue exclude staining, MTT assay, Giemsa staining, Hoechst 33324,AO/EB double fluorescent dye staining, Annexin V/PI, PI flow cytometry, DCFH-DA,Rhodamine 123 staining were used to detect and quantitate cell death and apoptosis.Reactive oxygen species and mitochondrial membrane potential were also used to investigate the protection mechanisms of quercetin on rotenone induced apoptosis.Following conclusions were demonstrated from this paper:1. The results of MTT assay and trypan blue staining showed that rotenone had a strong cytotoxicity, the growth inhibition effect were in time and dose-dependent manners. And quercetin had not cytotoxicity to cells when at 1 μM, 10 μM, 50 μM,but 10 μM quercetin in combination with rotenone can significantly improve the rotenone-induced cell viability decreased.2. The morphology of cells observed by the inverted microscope exhibited that treated cells by rotenone became round and shrunken, poor adherence, but quercetin have relief these characteristics induced by rotenone.3. The results of staining of Giemsa, Hoechst 33324 and double sequentialAO/EB staining showed that the representative characters of apoptosis chromatin condensation and nuclear fragmentation could be induced by rotenone, pretreated with quercetin the number of apoptotic cells was decreased.4. Annexin V/PI double staining assay and PI staining by flow cytometry reavealed that rotenone caused phosphatidylserine(PS) out leaflet and cell membrane permeability changes, appeared early and late apoptosis, cell cycle arrest at the G2 phase cycle. And when protected by the quercetin, cell apoptosis rate and G2 arrest were decreased.5. Flow cytometry assay and fluorescence microscopy observations by DCFH-DA and Rhodamine 123 staining suggest that the intracellular ROS and mitochondrial membrane potential were significant dependent by time-dose when cells were treated by rotenone for 3 h, 6 h, 12 h. In rotenone-treated cells the intracellular ROS levels is increased, the mitochondrial membrane potential is decreased compared with control.The pretreatment of quercetin can reduce the amount of ROS produced by rotenone,and improve rotenone-induced mitochondrial membrane potential reduce.In conclusion, Quercetin protected the rotenone-induced apoptosis in U87 through reducing ROS production, increased mitochondrial membrane potential.