Explore The Molecular Biological Mechanism Of Quercetin Inhibiting Glial Scar Healing

Quercetin, a flavonoid compound which was found widely distributed in many plants and vegetables has been confirmed to possess antihistamine, anti-inflammatory, antiviral, immunomodulatory and antioxidant properties. A large of studies have reported that quercetin performs an effective role in anti-oxidation, anti-inflammation, against cancer and protecting nerve cells. A large of studies has indicated quercetin can physiologically protect neurons and alleviate the progression of neurodegeneration diseases through altering related gene expression in astrocytes. Therefore, exploring the effect of quercetin on astrocytes scratch healing and its molecular mechanismit 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.Glia maturation factor, a17-kDa protein which was made up of142amino acid residues, mainly includes glia maturation factor-β(GMFB) and glia maturation factor-y (GMFG). GMFB detected predominantly in the vertebrate brain was initially identified as a growth and differentiation factor acting on neurons as well as glia. The gene and amino acid sequence of GMFG is highly homologous to GMFB (71%identity with the GMFB gene and78.9%identity with the GMFB amino acid sequence in rat), so it was named glia maturation factor-γ, a novel member of the GMF gene family. In contrast to the distribution of GMFB, GMFG was predominantly detected in thymus and to a lesser extent in brain. The expression distribution pattern of the GMFG differs from that of the GMFB gene. The GMFG gene is highly expressed in blood (including in myeloid leukemia and lymphoid leukemia cell lines), thymus, spleen, fetal liver and lung. GMFG protein coevolved with increasing complexity of the blood/immune system retaining proliferative and/or differentiative potential. GMFG gene in nervous/brain tissue is not higher than that in any other tissue, in rat brain, mRNA of GMFG was localized around pyramidal cells within CA3of hippocampus. GMFG observed also in the inner limiting membrane from E14to P1rat retina may contribute to the development and growth of glia and neurons. But to date, the distribution of expression and overall function of GMFG in brain remains elusive.In recent years, more attentions focus on its antitumor activity which can induce apoptosis of various tumor cells, such as leukemic cells, pancreatic tumor and so on. Oral squamous carcinoma, are the sixth most common malignancy worldwide. Oral surgery combined with radiotherapy or chemotherapy is one of the optimal treatments. However, multidrug resistance (MDR) gives the cells resistant to chemotherapeutic drugs and resulted in chemotherapy failure. One of mechanisms involved MDR is the expression of P-glycoprotein (P-gp). Many studies show that quercetin can inhibit p-glycoprotein expression to decrease the drug resistance in a variety of cancers, such as Human Pancreatic Carcinoma and Caco-2cell mono layers. Whether or not quercetin inhibited Oral squamous carcinoma growth or reversal multidrug resistance were archived through inhibiting P-gp expression is not yet clear.Our study is divided into three parts as below: Part one Explore the molecular biological mechanism of quercetin inhibiting glial scar healingOBJECTIVEFirstly, we established an in vitro migration model of rat astrocytes and studied the effects of quercetin on the migration velocity of astrocytes. The present study aims to investigate the effect of quercetin on the migration and proliferation of astrocytes in glia scar, as well as the probable mechanism, in an effort to identify an effective drug as a potential candidate for treating glia scar.METHODS1. Immunofluorescence The generation of primary astrocytes was identified by GFAP fluorescence, positive cells of more than95%, in line with the requirements.2. Establishment of astrocytes migration model in vitro Firstly, we established an in vitro migration model of rat astrocytes and studied the effects of quercetin on the migration of astrocytes. Primary cultured astrocytes were divided into normal control group(C) with only scratching treatment, DMSO group (D) with scratching treatment and quercetin dissolved in DMSO group (Q) with scratching treatment. Total18crisscross scratches were done with a sterile white pipette tip in35mm Petri dishes, and then the three groups of cells were subjected to incubation at37℃5%CO2. Individual images were taken with the10×magnification lens, and the cell-free width was measured using NIS Elements D software to calculate the cell-free index. The cell-free index was calculated according to the Calculation Formula (Ix=3Lx/(Lc+Ld+Lq)). Through the statistical analysis effect of quercetin on the migration of astrocytes was further explored.3. Western Blotting In order to further examine the effect of quercetin on caspase-3the ERK1/2and phosphorylation level, western blotting were performed to examine expression of caspase-3, ERK1/2and p-ERKl/2.The pictures were quantified by Gel-Pro analyzer (Alpha Innotech Corp, CA), and protein levels were expressed as the relative values of the controls.4. Live/Dead Staining Cultivating mature astrocytes which were washed several times with PBS were incubated for15min with an appropriate mixture of the SYTO9and propidium iodide stains at room temperature. After several washes with PBS, the photos were taken with confocal images. Astrocytes with intact cell membranes stained fluorescent green, whereas cell with damaged membranes stained fluorescent red.5. Click-iT EdU Test In order to examine effect of quercetin (50μmol/L) on proliferation of astrocytes and explore the mechanism, Click-iT EdU Test was performed. Half of medium for astrocyte cells were replaced with fresh media containing20μmol/L of EdU. After incubation about24h, the cells were fixed by lmL of3.7%formaldehyde in PBS for15min at room temperature. The permeabilization was carried out by1mL of0.5%Triton X-100in PBS. After washing with1mL of3%BSA in PBS,0.5mL of Click-iT reaction cocktail was added to each well. The samples were kept from light for30min at room temperature. After washing with1mL of3%BSA in PBS, the cells were mixed with1mL of1X Hoechst33342solution for30min, and then proceed to imaging and analysis.6. Flow cytometry The cells were centrifuged at1500r/min for5min and resuspend with250μL of trypsin buffer. After10min,200μL of trypsin inhibitor and RNase buffer was added and mixed with200μl of cold propidium iodide stain solution. The samples were gently mixed and incubate for10min in the dark on ice. The analysis will be carried out by the flow cytometry within3hours.RESULTS1. Effect of quercetin on the closure of scratches in vitro Through the statistical analysis we found that quercetin(50μmol/L) could inhibit the closure of scratches in vitro in comparison with C and D group, however DMSO treatment has no effect on the closure of scratches(data were not shown), at6h there was a statistically significant differences. At72h part of the vision in group C and D group have healed, so their widths were not measured.2. Analysis of astrocytes viability Live/dead dyeing showed that there was no statistically significant difference in quercetin treatment (50μmol/L) group compared with C and D group. Quercetin had no effect on astrocytes viability. We inferred that the inhibited scratch closure was not due to interfered with astrocytes viability.3. Effect of quercetin on astrocytes apoptosis Immunocytochemistry analysis utilizing anti-caspase-3antibodies revealed that the treatment of quercetin (50μmol/L), didn’t lead to expressing change of caspase-3. Western blotting also didn’t examine expressing change of caspase-3(32kDa) in the treatment of quercetin.Quercetin had no effect on apoptosis induction of astrocytes.4. Effect of quercetin on astrocytes proliferation and analysis of cells cycle Through the Click-iT EdU Test and flow cytometry analysis we found there were statistically significant differences in quercetin treatment group compared with C and D group. The proliferation ratio of astrocytes in control group was ten times than the treatment groups. When compared with control, quercetin increased the pop pulation in the G1phase from82.04%to92.06%at24h, with a corresponding decrease in the S and G2phase. This data corroborates the potent inhibitory effect of quercetin on DNA synthesis.5. Quercetin inhibits the migration of astrocyte through affecting phosphorylation of ERK1/2Quercetin (50μmol/L) decreased the p-ERKl/2of astrocytes about45%at24h compared with control. The change of ERK1/2phosphorylation level had a certain time and concentration-responses. At12h the phosphorylation levels of ERK1/2is the lowest in time-response curves. Along with the increase of the quercetin concentration (0-100μmol/L), the ERK1/2phosphorylation level gradually decrease, at100μmol/L the phosphorylation levels of ERK1/2is the lowest in concentration-response curves By contrast, U0126significantly retarded wound closure. By maintaining ERK1/2activation with the phosphatase inhibitor vanadate, we found maintenance of ERK1/2activation with vanadate restored migration in quercetin-treated cells thus supporting that ERK1/2governed directional movement in astrocytes.CONCLUSIONSIn this study we found there were no effect of quercetin (50μmol/L) on survival and apoptosis induction of astrocytes and quercetin inhibited glial scar formation through inhibiting migration and proliferation of astrocytes in glia scar. Part two The Analysis of Glia Maturation Factor Expression in Rat Brain and Glia Maturation Factor difference expression between astrocyte and glioma cell contributes to the pathogenesis of astrocytomasOBJECTIVEExamine whether GMFG protein is expressed in rat brain. Besides, we made a analysis of the cell/tissue distribution of expression between GMEB and GMFG and a preliminary study of GMFG function in the astrocyte.METHODS1. Cell culturePrimary astrocytes culture, Primary neurons culture, Glioma9L Cell and Glioma C6Cell lines culture2. Glioma Tumor Samples and Tissue Dissection All tissue samples and clinical information were collected at the Department of Neurosurgery, Nan fang Hospital, SMU, Guang zhou.3. Immunofluorescence Cellular immune fluorescence staining was used to examine whether GMFB and GMFG expressed in primary astrocytes.4. Western Blotting Western Blotting was performed to examine whether GMFB and GMFG expressed on primary astrocytes and rat brain.5. Immunohistochemistry Staining Immunohistochemistry Staining was used to examine distribution and expression of GMFB and GMFG in CNS.6. RNA Isolation and Real-time PCR RNA Isolation and Real-time PCR were used to perform to examine the expression differences of GMFB and GMFG between in primary astrocytes and glioma cell lines in mRNA level.7. Immunoprecipitation Immunoprecipitation was used to explore the interation between GFAP and GMFB/GMFG in CNS.RESULTS1. Expression of Rat GMFB and GMFG in the central nervous system For the first time, in this study we proved the existence of GMFG in the central nervous system at the level of gene, protein and cell. By Immunofluorescence used polyclonal anti-rat GMFB and GMFG antibodies we found that GMFB expressed in all astrocytes and mainly located in cell nucleus of astrocytes, while GMFG only expressed in a part of astrocytes and located in cell nucleus and cytoplasm where expression of GMFG present stellation.2. The tissue region-response distribution of GMEB and GMFG in rat brain The specific anti-GMFB antibody visualized the bands in the whole brain and predominantly in pyramidal cells of hippocamp. In contrast, the specific anti-GMFG antibody wasn’t detected in the whole brain, GMFG was positive only in a part of astrocytes; GMFG is predominantly detected in pyramidal cells of hippocamp and to a lesser extent in cerebral medulla and basal ganglia (cerebram), cerebral cortex is the weakest.3. The age-responses expression of GMEB and GMFG in rat brain The level of GMFB mRNA reached a peak at D7and thereafter becomes obviously reduced; while GMFG mRNA persisted in reducing until older. By western blotting analysis, GMFB were detected in rat brain every age phase after birth and persisted until older; GMFB protein reached the peak in the seventh days after birth with decreasing after that and plateauing from the second week to the third month after birth; while GMFG protein persisted in reducing from the first day to the eighth month after birth with minor4. The analysis of GMFB between astrocyte and glioma cells Our data show that nuclear expression of GMFB in rat glioma cells increased, but there was no obviously change in cytoplasmic GMFB. The former results show that as the central nervous system gradually matured, the expression of GMFB in astrocytes gradually decreased. Glioma cells as a immature and undifferentiated glial cells presented higher expression of GMFB in the nucleus. So we hypothesized that there may be negative feedback control system between the regulation of GMFB and maturity degree of astrocyte. It may be this negative feedback control mechanism that causes the increase of GMFB in the nucleus of rat glioma9L cells and glioma C6cells.5. The analysis of GMFG between astrocyte and glioma cells Our study show that the expression of GMFG in glioma significantly reduced compared to that of astrocyte in the mRNA and protein level. And by Immunofluorescence analysis we found that fluorescence intensity of GMFG in rat glioma cells is obviously weaker than that of astrocytes. Our results showed that there was some kind of interaction between GMFG and GFAP, while there was no this kind of interaction between GMFB and GFAP.6. The positive correlation analysis between expression of GMFG and the glioma GMFG expression was observed in4/5(80%) of normal brain,4/7(57.14%) grade Ⅳ glioma. The positive rate of GMFG was significantly lower in grade Ⅳ glioma than that of normal brain (p<0.05,χ2=6.5, Fisher exact test). GMFG was positive in the cytoplasm and nuclei of cortical. In positive cases, the cells, regardless of the types, showed the cytoplasm and nuclei immunoreactivity for GMFG. In order to further confirm this conclusion, Western blot show that the expression of GMFG in grade Ⅳ astrocytomas significantly reduced compared to that of astrocyte in the normal brain tissue.CONCLUSIONSFor the first time, in this study we proved the existence of GMFG in the central nervous system at the level of gene, protein and cell. And then we compared the tissue distribution of expression between GMEB and GMFG in rat astrocyte and neuron and found their tissue region-response distribution and age-response expression in rat brain. In addition, we found the difference in expression of GMFB and GMFG between glioma cell and astrocyte. For the first time, we put forward the hypothesis that there was a negative feedback control mechanism regulating expression of GMFB in the glial cell and made a preliminary study of GMFG function in he pathogenesis of astrocytomas. Part three Effect of quercetin on KBV cells and its resistance mechanismOBGECTTVEHere we investigated the effects of quercetin on migration, invasion, proliferation and apoptosis of the oral cancer cell lines KBV which were vincristine (VCR)-resistant. Furthermore, we assessed the sensitivity of KBV cells to VCR by inhibiting the expression of P-gp with quercetin.METHODS1. Cell culture KBV cells were cultured in RPMI-1640culture media containing10%fetal calf serum and200nmol/L VCR at37℃with5%CO2.2. In vitro migration assay KBV cells (5×104cells) were placed in the upper compartment, and200μL RPMI-1640culture media containing10%fetal calf serum and200nmol/L VCR was added to the lower compartment. The transwell plates were incubated at37℃for12h in a humidified atmosphere with5%CO2and stained with10%crystal violet. Migration cells were defined as cells that had moved into the lower surface of the membrane, and the non-migrating cells which were retained on the upper surface of the membrane were removed by a cotton swab.3. In vitro invasion assay Briefly, transwell units were coated with150-200μL/cm2Matrigel at room temperature for30min to form a genuinely reconstituted basement membrane. KBV cells (5x104cells) were placed in the upper compartment, and500μL RPMI-1640culture media containing10%fetal calf serum and200nmol/L VCR was added to the lower compartment. The transwell plates were incubated at37℃for36h in a humidified atmosphere with5%CO2and stained with10%crystal violet. Invading cells were defined as cells that had degraded the Matrigel and moved into the lower surface of the membrane, and the non-invading cells which were retained on the upper surface of the membrane were removed by a cotton swab.4. MTT assay KBV cells at the logarithmic phase were collected, incubated in a96-well plate at a concentration of5×103cells and cultured for24h. Following the attachment of the cells to the wall, RPMI-1640medium containing quercetin (0,25,50,75or100μmol/L) were supplemented at a final dose of200μL/well. Following12,24and48h of culture,15μL of lg/L MTT solution was added and the cells were cultured for4h. The supernatant was then removed, and150μL DMSO was added to each well and slightly shaken for15min. The optical density (OD) value was detected at a wavelength of490nm by Microplate Reader. Six duplicates were designed for each well, and the mean value was calculated three times.5. Click-iT EdU Test Half of medium for KBV cells were replaced with fresh media containing20μmol/L of EdU. After incubation about24h, the cells were fixed by lmL of3.7%formaldehyde in PBS for15min at room temperature. The permeabilization was carried out by1mL of0.5%Triton X-100in PBS. After washing with1mL of3%BSA in PBS,0.5mL of Click-iT reaction cocktail was added to each well. The samples were kept from light for30min at room temperature. After washing with1mL of3%BSA in PBS, the cells were mixed with1mL of1X Hoechst33342solution for30min, and then proceed to imaging and analysis.6. The cell cycle analysis by Flow cytometry The cells were centrifuged at400g for5min and resuspend with250μL of trypsin buffer. After10min,200μL of trypsin inhibitor and RNase buffer was added and mixed with200μL of cold propidium iodide stain solution. The samples were gently mixed and incubate for10min in the dark on ice. The analysis will be carried out by the flow cytometry within3hours.7. Apoptosis assays by Flow cytometry Briefly, cells (1×106per ml) were washed in PBS and suspended in500μL binding buffer. Annexin V FITC (5μL) and propidium iodide (5μL) were added to the cells for10-15min in the dark at room temperature. The analysis will be carried out by the flow cytometry within1hour.8. Western Blotting In order to further examine the effect of quercetin on caspase-3, Bax, Bcl-2and P-gp, western blotting were performed to examine expression of caspase-3, Bax, Bcl-2and P-gp. The pictures were quantified by Gel-Pro analyzer (Alpha Innotech Corp, CA), and protein levels were expressed as the relative values of the controls. RESULTS1. Analysis of viability in KBV cells To investigate the inhibitory effect of quercetin-induced KBV cells viability, KBV cells were treated with various concentrations of quercetin. The growth inhibition rate increased in a dose and time dependent manner. Following treatment with low-dose quercetin (25μmol/L) for12,24and48h, the growth inhibition rates of the KBV cells were (13.76±2.679)%,(14.63±4.262)%and (16.80±3.876)%respectively. And no any differences were observed in these groups. However, Quercetin gradually decreased the KBV cells viability when quercetin does at50μmol/L for48h or more than this dose, such as75μmol/L for12to48h or100μmol/L for12to48h. The viable cells were about50%under treatment with75μmol/L concentration of quercetin for48h. To better reflect its pharmacological effects, the50μmol/L of quercetin doses was selected for next studies.2. Quercetin inhibited migration and invasion of KBV cells The migration and invasion of KBV cells were significantly inhibited by quercetin at or more than25μmol/L compared with cells treated with DMSO. Quercetin at25,50and100μmol/L inhibited the migration to60.14%、30.40%and13.93%and inhibited the invasion to38.57%、22.27%and5.12%compared with control which considered as100%.3. Effect of quercetin on KBV cells proliferation and cells cycle The percentage in cells proliferation was significant lower when quercetin treatment applied. The proliferation rate of KBV cells in control group was two times than the quercetin treatment groups. Flow cytometry results showed that quercetin at50μmol/L treatment for24h increased the population in the G1phase from42.41%to52.66%(P<0.01), and with a corresponding decrease from52.24%to43.04%in the S phase (P<0.01). 4. Quercetin induced KBV cell apoptosis Comparing with untreated KBV cells, the apoptosis proportion in quercetin-treated KBV cells were significantly increased. The percentage of apoptotic cells increased in a dose-related manner, reaching a17.53%value when quercetin at100μmol/L.5. Expression of Bcl-2, Bax and caspase-3protein in KBV cells The Bcl-2protein levels and the35kDa pieces of caspase-3decreased in a dose-dependent manner in cells treated with quercetin for24h, whereas the Bax protein levels and17kDa fragments of cleaved caspase-3increased in a dose-dependent manner. Those data showed that quercetin-induced apoptosis of KBV cells is probably mediated by the mitochondrial pathway.6. The effect of quercetin on P-glycoprotein expression Incubate KBV cells with quercetin at0,25,50,75and100μmol/L for24h, and examine the changes in P-gp protein. The results of the Western blotting revealed that the protein level of P-gp was significantly decreased in quercetin treatment KBV cells. The protein levels decreased in a dose-dependent manner. Quercetin at25,50,75and100μmol/L decreased P-gp to91%,83%,66%and45%, respectively, as compared with the control.7. Quercetin enhances the sensitivity of KBV cells to Vincristine by down-regulating P-glycoprotein expression The relative levels of p-gp in cells treated with the combination of VCR and quercetin were significantly lower than those treated with VCR alone and control. Meanwhile, the proliferation rate in KBVcells treated with VCR and quercetin significantly decreased and the apoptosis proportions were significantly higher than that in other groups. The migration rate and invasion rate were also decreased to8.82%and12.7%respectively. Those data provides ample evidence that quercetin enhances the sensitivity of KBV oral cancer cell lines to VCR by down-regulating P-gp expression. CONCLUSIONSThis study provided evidence that quercetin inhibited the migration, invasion, proliferation and signaling molecules involved in anti-apoptosis and drug resistance of KBV oral cancer cell lines and enhanced its sensitivity to Vincristine...