The Effects Of Fluoride On Nrf2-ARE Signal Pathway In Rat Osteoblasts

BACKGROUND AND OBJECTIVEAs we know, fluorine is the indispensable trace element for the humans, it is very important for skeleton growth and development and it can maintain skeleton physiological structure. But at the excessive condition, fluoride can damage bone system which is clinically characterized in humans by dental and skeleton fluorosis. In recent years, it has been demonstrated that the prevention and treatment measure of Enamel fluorosis have been got great of success, but it generates serious influence to people'healthy who living in the areas. So, go deep into the definite pathogenesis of fluoride poisoning has significance in prevention and treatment of the health damage by Enamel fluorosis. At present, although the molecular mechanisms for fluoride poisoning have not yet been fully clarified, it has been suggested that apoptosis induced by oxidative stress is a critical mechanism in many mechanisms of chronic Fluorosis. Chronic fluoride exposure can cause oxidative stress in vivo and generate excessive reactive oxygen species (ROS), while low concentrations of ROS act as an intracellular signal reaction between transfersomes and play a pivotal role via some of the body-mediated oxidative stress-sensitive signal transduction pathways. In resent years, the nuclear transcription factor (transcription factor NF-E2-related factor 2, Nrf2) and a substrate linker protein (Kelch-like ECH-associated protein, keap1) are center modulates in antioxidant defense, So Nrf2 is being more and more attended in the role of regulation of oxidative stress. The nuclear transcription factor Nrf2 forms heterodimers with other bZIP transcription factors and then binds to the antioxidant response element (ARE) to mediate the transcriptional up-regulation of phase 2 gene and code the expression of antioxidant enzymes and phase 2 drug metabolizing enzymes in cytoprotection, increased the body resistance and maintaining the body to survive.Nrf2-ARE signal pathway plays an important role in antioxidant defense through activated the antioxidant mechanism and restrains the inflammation signal pathways. For example, heme oxygenase 1(HO-1) and quinone oxidoreductase 1(NQO1) expression induced by Nrf2, which may play a critical role in enhance the body of antioxidant capacity. In summary, this research used normal culture rat osteoblasts to expose NaF in vitro, studied the effects of NaF on oxidant stress, the expression of Nrf2, HO-1 and NQO1 rnRNA, the cell proliferation and apoptosis and explored the effects of fluoride on Nrf2-ARE signal pathway in rat osteoblasts.METHODS1 Experimental methods1.1 Cell culture and identificationPrimary osteoblasts were derived from bone (calvaria) of neonatal Sprague-Dawley rats that were maintained at Center of Laboratory Animal of Guangzhou University of Chinese Medicine. Primary cells were isolated from parietal calvaria from which the periosteum and cranial sutures were removed to reduce non-osteoblast cell contamination. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and incubated at 37°C in a 5% CO2 atmosphere with a change of media every three days until they reached 80% confluency. Cells were subcultured to 1:2 for passage under identical conditions for utilization in experiments. The cells via morphological observation, NBT/BCIP stained of appraisal and draw cell growth curve.1.2 Cell morphological observation and proliferation detectionOsteoblasts were seeded in 6-well plates and incubated in complete DMEM containing NaF for 24h, then observed the influence of fluoride on cell structure and photographed. Osteoblasts were seeded on 96-well plates and incubated in complete DMEM containing NaF for 24h, 48h and 72h, osteoblasts cell viability was measured by MTT.1.3 Cell cycle and apoptosis assayOsteoblasts were seeded in 6-well plates and cultured for 24h, then incubated with NaF for 24h,48h and 72h, the cells were collected, cells cycle and apoptosis rates of osteoblasts were evaluated by Annexin V/PI apoptosis Kit(Ex=488nm, Em=530nm) though flow cytometry(FCM). Used MULTYCYCLE software analyse the cell cycle and the WINMDI software for the analysis of apoptosis rates.1.4 The detection of the level of oxidative stressOsteoblasts were seeded in 24-well plates, the activities of glutathione peroxidase (GSH-Px), SuperoxideDismutase (SOD) and the contents of malondinaldehyde (MDA) in the supernatants of the cells were detected by biochemical methods separately.1.5 The detection of the DNA damageThe single cell gel electrophoresis(SCGE)was used to measure the DNA damage of osteoblasts : used the inverted microscope observe the modify of cellular morphology and the image analysis IMI1.0 analyze each of cells. The Elisa was employed to measure the level of 8-OHdG: osteoblasts were seeded and incubated with NaF for 24h, 48h and 72h, used 8-OHdG kit according to the manufacturer's to measure the contents of 8-OHdG.1.6 RNA isolation and real-time PCROsteoblasts were seeded in 10cm dishes, experimental group as follows: NaF group and tBHQ pretreatment group. Total RNA was isolated using TRIzol reagent, the mRNA levels of HO-1, NQO1 and Nrf2 were quantified by real-time quantitative PCR, used PrimeScriptTM RT-PCR Kit.1.7 Nuclear extraction and western blot for Nrf2Osteoblasts were seeded in 10cm dishes, experimental group as follows: NaF group and tBHQ pretreatment group. Cytoplasm and nuclear protein was extraction by NucbusterTM Protein Extraction Kit, the protein quantification was measure by the BCA Kit. Used semi-dry transfer method to transfer protein to PVDF membrane (250mA,80min). The membrane was blocked in 5% non-fat milk in tris-buffered saline containing 0.05% Tween 20(TBST), then it probed with a primary antibody (1:300 for Nrf2, 1:500 for -actin), Membrane was washed repeatedly in TBST before being probed with secondary antibody (1:2000). Following repeated washes, visualization of the protein-antibody conjugate was performed used enhanced chemiluminescence.2 Statistical analysisData were expressed as mean±S.D. Differences between groups were assessed with one-way ANOVA or student's t-test with SAS 9.0, according to data feature. Differences were determined to be significant when P<0.05, significant level a=0.05. RESULTS1 Cell culture and identificationOsteoblasts isolated initially from rat calvarial bone were in tiny spherical shape, have strong bright refractive which were observed under inverted microscope. Osteoblasts show the main spindle, slightness spindle, triangle shape and an irregular shape after adherence to culture flask. Cells were identified as osteoblasts via morphological observation and NBT/BCIP stained.2 Effects of NaF on morphology and proliferation of osteoblastsOsteoblasts were incubated with various concentration of NaF, the control group cells were in single-layer, overlapping growth and gathered clouds of proliferation status. There is a slightly reduced of the numbers of osteoblasts in low doses, while in high doses the cells were significantly reduced, intercellular and empty bubble also increased. The proliferation activities were restrained and the cell survival was a declining trend with the concentration of NaF increase and the incubated time elongation.3 Effects of NaF on the cell cycle and the cell apoptosisThe effect of NaF on the cell cycle was that the number of cells at G0/G1 phase was increased, at the same time decreased the number of the cells at S and G2/M phase. There was no change on apoptotic rate in 0.25,0.50,1.00,2.00mmol/L NaF, however significantly higher apoptotic rate was found in the 4.00mmol/L NaF when compared to the control group.4 Effects of NaF on the active antioxidant enzymes of osteoblastsCompared with the control group, the activities of SOD and GSH-PX was decreased in different concentration of fluoride at 24h and 48h, while in 72h the activities of SOD was increased first and then decreased. The level of MDA was increased along with different concentration of NaF ( P<0.05) from 24h to 72h.5 Effects of NaF on the DNA damage of osteoblastsThere was no signifinance on DNA damage for 24h, osteoblasts were incubated with the concentration of 0.25~4.00 mmol/L NaF for 48h and 72h , Compared to the control group , the general indices(tail rate, tail length, olive tail moment, tail moment, DNA content in tail)were increased in the concentration of 0.25~2.00 mmol/L NaF, difference were statistically significant(P<0.05). But when osteoblasts were exposed to NaF 4.00 mmol/L, the general indices were decreased compared to the other concentration. The content of 8-OHdG was an increased trend along with different concentration of NaF.6 Effects of NaF on the gene expression and the content protein of Nrf2NaF group: after exposure to NaF for 24h and 48h, NaF can continue to induce Nrf2 gene expression, however, there was no change on mRNA expression level of Nrf2 gene in osteoblasts with the time prolong to 72h. At 24h and 48h, NaF can decrease Nrf2 protein in cytoplasm. Nrf2 protein in nucleus was increased than the control group at 48h. tBHQ pretreatment group: tBHQ pretreatment of 12h and then exposure to NaF incubated for 24h, 48h and 72h, the level of Nrf2 gene expression was significantly lower than a separate group NaF group, however, the level of Nrf2 gene expression was significantly increased compared with the control group. The content of Nrf2 in nuclear protein was increased than a separate group NaF group at 24h.7 Effects of NaF on the HO-1 and NQO1 gene expressionNaF group: There was no change on mRNA expression level of HO-1 and NQO1 gene at both 24h and 72h. The mRNA expression level of HO-1 and NQO1 was significant increased in 0.25 mmol/L NaF (P<0.05), However, The mRNA expression level of HO-1 was significant decreased in 0.50, 2.00 and 4.00mmol/ L NaF, and the mRNA expression level of NQO-1 was significant decreased in 4.00 mmol/L NaF.tBHQ pretreatment group: tBHQ pretreatment of 12h and then exposure to NaF incubated for 24h and 72h, there was no change on HO-1 gene expression. However, tBHQ can induce HO-1 gene expression in 4.00 mmol/L NaF at 48h. The mRNA expression level of NQO-1 was significant decreased in 4.00 mmol/L NaF than NaF group at 24h, and NQO-1 gene expression was significant decrease in (0.25, 1.00 mmol/L) NaF than NaF group at 48h, the mRNA expression level of NQO-1 was significant decreased in (0.50 mmol/ L, 1.00mmol/L) NaF than NaF group at 72h.CONCLUSIONS1 NaF could restrain cell proliferation, change cell cycle, but only in high dosage (4.00mmol/L) can induced apoptosis.2 NaF could induce lipid peroxidation and DNA oxidative damage, as well as decrease the activities of antioxidative enzymes system in rat osteoblasts.3 NaF could induce Nrf2 gene expression and increase Nrf2 protein, leaded to overexpression of HO-1 and NQO1 to enhance the antioxidant abilities of cells.4 Under the condition of this study, tBHQ pretreatment could decrease the mRNA expression level of Nrf2, HO-1 and NQO1 and the content of Nrf2 protein.