Molecular Mechanism Of Effect Of Quercetin On Reactive Astrocytes

BACKGROUND AND OBJECTIVEAstrocytes are the most abundant cell in CNS, it has been divided into two main subtypes, protoplasmic and fibrous, on the basis of differences in their cellular morphologies and anatomical locations, the former are found throughout all grey matter, while the latter are found in all white matter. Both astrocytes perform very important physiological function in CNS, including maintenance of homeostasis, elimination of active reagents, supporting and nourishing in neuron regeneration, promoting formation and remodeling of branching processes of neurons.etc. Astrocytes become reactive and some changes such as cellular hypertrophy, synapses thinner and longer and alterations of gene expression in response to CNS injury of trauma, stroke, infection or degenerative disease. In response to sever and persisting injury, Reactive astrogliosis can lead to the appearance of newly proliferated astrocytes and migrate to the injury region, forming the glial scar with immunocytes and oligodendrocytes. In hence, we can conclude that CNS disorders is a complex polycellular reaction involved by a large body of intracellular and extracellular signal molecules, in which reactive astrocytes play either beneficial or detrimental roles. In multiple cerebral injury models, exploring the mechanism how reactive astrogliosis and glial scar get involved in cerebral ischemic hypoxia and seeking therefore an effective approach to prevent or treat related brain diseases is intriguing more and more scientists’curiosity.Quercetin is a flavonoid widely distributed in vegetables, fruits and plants. A vast of studies have reported that quercetin perform an effective role in anti-oxidation、anti-inflammation、against cancer、and protecting nerve cells. A vast of studies has indicated quercetin can physiologically protect neurons and alleviated the progression of neurodegeneration diseases through altering related gene expression in astrocytes. Therefore, we explore the molecular mechanism of gene expression alterations in astrocytes following quercetin stimulation; it will lay the foundation for further study of the roles of reactive astrocytes in cerebral injuries and cast new light on the study of glial scar formation.Our study is divided into three parts as below:Part one Effect of quercetin on gene expression in glucose-oxygen deprived astrocytes We use large-scale oligo microarray technology to study the difference of gene expression in glucose-oxygen deprived astrocytes after quercetin treatment, which are verified by real-time quantitative PCR.It is convenient for us to learn those genes having join the role of quercetin on reactive asrocytes processes,which provide theoretical basis for exploring molecular mechanism of the role of quercetin.Part two Modulation of quercetin on the expressions of AIF-1and Rac2in reactive astrocytesAccording the results of part one, we select AIF-1and Rac2genes from differently expressions of many genes. We build inflammatory and injury models to study the influence of quercetin on AIF and Rac2expression of primary astrocytes, indicating the role of AIF-1and Rac2in reactive astrocytes after inflammatory and injury stimulation and the role of quercetin on the inhibition of reactive astrogliosis. Part three Effect of quercetin on the growth and migration of glioma cellsWe previous found quercetin could inhibit reactive astrogliosis and migration. Glioblastomas are the most common malignant tumors that arise from astrocytic lineage, and are characterized by a high proliferation rate and aggressive invasion. They are not sensitive to radiation and chemotherapy, even resistant to many drugs. Our research discusses the influence of quercetin on the growth and migration of glioma, aiming to provide theoretical basis for molecular mechanism of glioma development and find effective therapeutic measures.METHODS AND RESULTSPart one Effect of quercetin on gene expression in glucose-oxygen deprived astrocytesMethods1. Separated and cultured primary astrocytes, then analyzed the purity of cultured cells through immunofluorescence staining.2. Built ischemic hypoxia model in vitro, and divided the cultured primary astrocytes into four groups:control,50μM quercetin, glucose-oxygen deprivation, glucose-oxygen deprivation plus50μM quercetin. Step:The medium were removed from C and C+Q50and added2ml10%FBS DMEM respectively, then cells were cultured in37℃5%CO2incubator for following4h.As to H and H+Q50, remove the medium, then add2ml DMEM without glucose and FBS, incubate in anaerobic (contain5%CO2+95%N2) incubator for4hs.Then displace the medium in C and H with2ml low glucose DMEM with10%FBS,while displace the medium in C+Q50and H+Q50with low glucose DMEM containing10%FBS and50μM quercetin, then simultaneously incubate the four groups in37℃5%CO2incubator for another24hs. 3. After the stimulation stated above, we detected the cell mortality and cytotoxicity of quercetin with LIVE/DEAD staining and LDH leaking rate methods.4. Applying large-scale oligo microarray to select the difference of gene expressions between H group and H+Q50group, and demonstrate the results by real-time quantitative PCRResults1. The generation of primary astrocytes was identified by GFAP fluorescence, positive cells of more than95%, in line with the requirements.2. Live/dead dyeing showed that the glucose-oxygen injury could clearly induce primary astrocytes injury comparing with the normal group. Most cells are dyed red and the shape of the green living cells also were changed obviously, including cell body hypertrophy, synapses thinner and longer. However, the count of dyeing red cells reduced, the count of green living cells increased and the injury cells decreased after glucose-oxygen deprived astrocytes treated with quercetin. In addition, quercetin didn’t change the shape and viability of primary astrocytes in normal condition.3. The release rate of LDH from glucose-oxygen deprived astrocytes Significantly increased from (8.24±0.6648)%to (54.60±1.6383)%, by6.63times(P<0.001). The release rate of LDH from injury astrocytes with50μM quercetin was (42.15±1.4671)%,which was less than glucose-oxygen deprived group(P<0.001).4. Total RNA was extracted from glucose-oxygen group and glucose-oxygen plus quercetin, which were used to analyze by Large-scale oligo microarray. It indicated the expressions of406genes had significant changed in mRNA level between the two groups. Upon further analysis of the informatics, repeated genes and EST clips were removed from those genes. And we ultimately ensured the different expression of180genes, of which49s were up-regulated and131were down-regulated. One hundred and forty-eight differentially expressed genes were confirmed by RT-PCR, including34up-regulated genes and114down-regulated genes, which showed that82.2%(148/180) genes that matched with the result of Large-scale oligo microarray.Part two Modulation of quercetin on the expressions of AIF-1and Rac2in reactive astrocytesMethods1.Cellular immune fluorescence staining was used to examine whether AIF-1and Rac2expressed on primary astrocytes.2.Primary astrocytes were stimulated by Pro-inflammatory cytokines IFN-γ10ng ml and TNF-alOng ml, IFN-γ lOng ml and TNF-alOng ml combined with either DMSO or querectin for24、48and72hours. Cells were harvested and subjected to analyzing AIF-1and Rac2expression by real-time quantitative PCR and western blot methods.3. Primary astrocytes were divided five groups:control,50μM quercetin, glucose-oxygen deprivation, glucose-oxygen deprivation combined with0.05%DMSO or50μM quercetin. The glucose-oxygen deprivation treatment were detailed in the part one methods. After treatment, Cells were harvested and subjected to analyzing AIF-1and Rac2expression by real-time quantitative PCR and western blot methods.4. Building the scratch wound model:(1). in order to make scratch wound uniformly, we prepared a35mm round card for damage template,which was painted14straight lines (seven by seven)by freely cross, spacing4.4mm between parallel straight lines.(2). put the card under the35mm culture dish, the monolayer cells were scratched straightly with a200μl pipette tip to create a wound.(3). after scratching, cells were washed three times with sterile PBS to remove floating cells and cell debris.(4).cells were respectively cultured with10%FBS DMEM,10%FBS DMEM containing0.05%DMSO or50μM quercetin for0h,24h,48h and72h.(5). contrast-phase photographs were taken by OLYMPUS IX71microscope to observe primary astrocytes migration, the relative migration area were measured by image pro-plus software.(6). after scratching wound, AIF-1and Rac2expression in primary astrocytes were measured by real-time quantitative PCR and western blot.Results1. We found AIF-1and Rac2express in the primary astrocytes cytoplasm by cellular immune fluorescence staining.2. Primary astrocytes were respectively stimulated24h,48h and72h by pro-inflammatory cytokines. qRT-PCR result suggested that pro-inflammatory cytokines could significantly increase AIF-1and Rac2expression (P<0.05); but50μM quercetin significantly reduced their expression, even less than control. However, AIF-1expression slightly increased by western blot analysis, quercetin had no effect on AIF-1expression. The expression of Rac2protein significantly increased after stimulating24h by INF-y and TNF-a, and increased more at72h, however, quercetin had an inhibition effect on pro-inflammatory cytokines-induced Rac2protein expression.3. qRT-PCR analysis showed quercetin significantly reduced AIF-1and Rac2expression in primary astrocytes with glucose-oxygen deprivation in vitro (P<0.001). We also found OGD injury could increase Rac2protein expression but AIF-1protein, quercetin only reduced Rac2protein level (P<0.01). 4. In order to study the effect of quercetin on primary astrocytes migration, we built scratch wound models in vitro. After scratching wound, primary astrocytes migrated to wound area in time-dependent,67.61%wound areas were covered at72h; DMSO group had a similar ability of migration to control. But quercetin significantly inhibited astrocytes migration, only53.13%wound area were covered(P<0.01).Moreover, we found scratch wound induced AIF-1and Rac2expression compare to Oh,and quercetin only decreased AIF-1mRNA rather than protein level, quercetin significantly decreased Rac2in both gene and protein levels.Part three Effect of quercetin on the growth and migration of glioma cells Methods1. Human U87glioma cells were seeded at5×103cells/well in10%FBS DMEM in96-well plates. After12h of incubation, the medium was removed and added new medium containing different concentrations of quercetin for24h,36h and48h. Control cultures were performed with10%FBS in the absence of quercetin. Cell viability was evaluated by3-(4,5-dimethylthiazol-2y1)-2,5-diphenyl tertrazolium bromide (MTT) assay.2. We used scratch wound models and transwell assay to evaluate migration of human U87glioma cells. Human U87glioma cells were seeded at2×105cells/ml in10%FBS DMEM.The detailed steps were seen in the method of part2. Migration assays were carried out using Boyden chambers.5×104cells were seeded onto the upper chamber in serum-free DMEM in the absence or presence of DMSO or quercetin. The lower compartment contained DMEM with10%FBS. After24h,the cells that migrated to the lower surface of the filter were fixed and stained with crystal violet and then counted in five random fields per well, and the extent of invasion was expressed as an average number of cells per microscopic field. Meanwhile, AIF-1and Rac2protein expressing in glioma cells with quercetin treatment or not were evaluated by western blot.Results1. MTT result showed quercetin inhibited the growth of human U87glioma cells in dose-dependent and time-dependent.2.We found that human U87glioma cells had significantly different migration ability between various treatments by scratch wound and transwell migration assay(P<0.001).Compared with control or DMSO group, cells with quercetin treatment migrated slowly, suggesting quercetin could inhibited human U87glioma cells migration.3. After scratching wound, AIF-1and Rac2expressions were increased, but quercetin significantly inhibited the expression of AIF-1and Rac2in glioma cells.INCLUSION1. Gene expression profiling by Large-scale oligo microarray analysis provides good understanding of the molecular mechanism of quercetin in glucose-oxygen deprived astrocytes, and laids the foundation for investigating the influence of quercetin and astrocytes in hypoxic-ischemic brain damage.2. AIF-1and Rac2expression in reactive astrocytes can exacerbate the progression of cerebropathy, while Quercetin can alleviate the development to some extent.3. Quercetin can inhibit the growth and migration of glioma cells.