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The Effects of Fluoride on Nrf2-ARE Signal Pathway in Rat Osteoblasts

Author: ZhongYuanFang
Tutor: YuRiAn
School: Guangdong College of Pharmacy
Course: Epidemiology and Biostatistics,
Keywords: NF-E2 -related factor 2 Heme oxygenase 1 Quinone peroxidase 1 Osteoblasts
CLC: R599
Type: Master's thesis
Year: 2011
Downloads: 131
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Abstract

Background and Objective Fluorine is one of the essential trace elements the body, on the whole body bone growth and maintenance of bone structure and function has an important physiological role. But the body will cause excess fluoride dental fluorosis and skeletal fluorosis psychosis phrenology damage. In recent years, although the prevention and treatment of endemic fluorosis has made great achievements, but still endemic fluorosis serious impact on the health of the people of the ward. Therefore, in-depth reveal the pathogenesis of fluorosis in endemic fluorosis caused health damage prevention aspect is important. At present, although the incidence of fluorosis molecular mechanism has not been fully elucidated, but the fluoride-induced oxidative stress-mediated apoptosis is still considered key to the formation of chronic fluorosis one of the mechanisms. Fluorosis occurs when the body can cause oxidative stress causes the body to ROS (Reactive oxygen species, ROS) increased, the low concentration of ROS as intracellular signaling intermediate transfer body reaction, mediated through oxidative stress in vivo sensitivity to certain signal transduction pathway plays an important role. In recent years, studies have found that the nuclear transcription factor NF-E2-related factor 2 (Nuclear factor E2-related factor 2, Nrf2) and its cytoplasmic adapter protein (Kelch 1ike ECH associated protein 1, Keap1) is the cell against oxidative stress central regulator of oxidative stress in this regard that the role played by more and more attention. Nrf2 be combined with other bZIP transcription factors form heterodimers then with antioxidant response element (Antioxidant response element, ARE) to be able to adjust and coding combined antioxidant enzymes and phase Ⅱ expression of detoxification enzymes, increased cell against oxidative stress resistance role in maintaining the body's survival. Nrf2-ARE antioxidant by activating cellular mechanisms of inflammation and inhibition of cell signaling pathways in the body against oxidative stress plays an important role. When heme oxygenase -1 (heme oxygenase-1, HO-1) and quinone oxidoreductase 1 (NADP (H): quinone oxidoreductase 1, NQO1) induced by Nrf2, can enhance the body's antioxidant capacity. In this study, primary cultures of rat osteoblasts to study fluoride on osteoblasts oxidative stress, DNA damage, cell proliferation and apoptosis, HO-1 and NQO1 gene expression and Nrf2 gene expression and its protein content explore the Nrf2-ARE signaling pathway in osteoblasts induced by fluoride damage in rats. Method 1 Experimental 1.1 osteoblasts culture and identification of newborn (1 ~ 2d) SPF level SD rats were purchased from Guangdong Province, Guangzhou University of Chinese Medicine Laboratory Animal Center. Rats were isolated under sterile conditions calvaria osteoblasts were the primary culture, the cells were cultured in DMEM medium (containing 15? S, 200 ∪ / ml double-antibody), 5% CO2, 37 ℃ under the conditions of cultivation. When cells reached 80% to 90% confluence, and passaged 1:2. Cultured cells by morphological observation, NBT / BCIP staining identified, and draw the cell growth curve. 1.2 osteoblast morphology and cell proliferation assay Osteoblasts were seeded in 6-well plates were observed fluoride on osteoblast cell morphology and radiography; the same number of osteoblasts were seeded in 96-well plates, cultured 24h, at a final concentration of NaF 0.25,0.50,1.00,2.00,4.00 mmol / L for exposure and continued to train 24h, 48h and 72h, MTT assay was used to detect cell proliferation. 1.3 cell cycle and apoptosis detection, cells were seeded in 6-well plates after 24h culture stained fluorine 24h, 48h and 72h, respectively, collected 12,000 cells by flow cytometry at an excitation wavelength Ex = 488nm, emission wavelength Em = 530nm to analyze the histogram of PI fluorescence. The United States PHEONIX's MULTYCYCLE software for cell cycle analysis using WINMDI software for the analysis of apoptosis. 1.4 osteoblasts measured levels of oxidative stress, cells were seeded in 24-well plates after 24h culture stained fluorine 24h, 48h and 72h, cells were collected, using nitrite determination of superoxide dismutase (SOD) activity, using dithiobis nitrobenzoic acid (DTNB) method for the determination of glutathione peroxidase (GSH-Px) activity, thiobarbituric acid (TBA) colorimetric method measuring lipid peroxidation product malondialdehyde ( MDA) content. 1.5 osteoblast DNA damage was determined by single cell gel electrophoresis (SCGE) detection of DNA tailing osteoblasts and injury: Preparation of agar slides under a fluorescence microscope observation and radiography, image analysis using SCGE System IMI1.0 individually analyze cell; using ELISA test (ELISA) detection of intracellular oxidative DNA damage product 8 - hydroxyquinoline deoxyguanosine (8-OHdG) content: cells were inoculated and infected 24h, 48h and 72h after collection The resulting cells, using an 8 - OHdG detection kit and microplate detection of intracellular 8-OHdG levels. 1.6 RNA extraction and RT-PCR experiments, cells were seeded in 10cm dishes were divided into: pure dye fluoride group, tBHQ pretreatment group. Extracted using Trizol reagent kit osteoblasts total RNA, using PrimeScriptTM RT-PCR Kit pure dye kit to detect and tBHQ pretreatment fluoride group consisting of bone cells HO-1 mRNA and NQO1 mRNA and nuclear transcription factor Nrf2 mRNA expression. 1.7 Western blot (western blotting) experiments, cells were seeded in 10cm dishes were divided into: pure dye fluoride group, tBHQ pretreatment group. NucbusterTM Protein Extraction Kit with extraction kit nucleoprotein and plasma protein, BCA protein quantitation kit for protein content, polyacrylamide gel electrophoresis, the protein transfer method using wet transferred to PVDF membrane (250mA, 80min). With 5% skim milk for the PVDF membrane was incubated with an anti-Nrf2 (1:300) and the internal reference?-Actin (1:500), the secondary antibody was 1:2000 concentration both hybridization light ECL kit according to the procedure chemiluminescence reaction, and its exposure imaging, imaging analysis system using gel gray value for protein analysis, the results with the experimental group and control group, the average gray value of the relative value. 2 Statistical analysis of data in each experimental group were expressed as mean ± standard deviation (x ± s) that the use of SAS9.0 statistical software for analysis. Meet the normal distribution and homogeneity of variance of the difference between the two groups was used to compare two independent samples t-test, comparing the differences between multiple groups using single factor analysis of variance, pairwise comparisons between groups using SNK test method, data When normality test is not satisfied with the non-parametric test, significance level ɑ = 0.05, P lt; 0.05 indicates significant difference. Results 1 cell culture and identification of freshly isolated cells was observed under an inverted microscope tiny translucent spherical refraction strong, adherent cells are mainly short fusiform, elongated fusiform, triangular and irregular shape. Cells by morphological observation and NBT / BCIP staining identified as osteoblasts. 2 fluoride on osteoblast cell morphology and cell proliferative activity of cells have been merged into the control group showed a dense monolayer, overlapping growth, gathered into a group of proliferative state, low-dose group decreased the number of cells and a small amount of cell vacuoles high-dose group showed significantly reduced the number of cells, cell gap increases, vacuoles increased. As the fluoride concentration and the fluoride time, the proliferation of osteoblasts were inhibited, cell viability decreased (P lt; 0.05). 3 of fluoride on osteoblast cell cycle and apoptosis of fluoride on osteoblast cell cycle mainly G0/G1 phase cells increased in order to facilitate, S phase and G2 / M phase cells decreased; 0.25,0.50,1.00,2.00 mmol / L dose group, apoptosis was not obvious, the difference was not statistically significant. 4.00mmol / L dose of fluoride on apoptosis induced significant apoptosis rate was significantly higher (P lt; 0.05). 4 of fluoride on oxidative stress in osteoblasts exposed to fluoride indexes 24h and 48h segment, compared with the control group, with the fluoride concentration increases osteoblast major intracellular antioxidant enzymes SOD and GSH-Px activity there is a downward trend But 72h segment, SOD activity decreased and then increased; and each time the high dose group of lipid peroxidation product MDA content was significantly higher than the control group (P lt; 0.05). 5 of fluoride on osteoblasts exposed to fluoride DNA damage 24h, osteoblasts DNA damage is not obvious, the fluoride 48h, 72h after each dose group of cellular DNA tail length, Olive tail moment, tail DNA% and tail / head length increase compared with the control group than that, and with increasing concentrations gradually increased, and the difference was statistically significant (P lt; 0.05), but 4.00mmol / L dose group, the index decreased compared to the previous dose. Intracellular oxidative DNA damage product 8-OHdG levels increased as the fluoride concentration was increasing trend (P lt; 0.05). 6 of fluoride on osteoblasts Nrf2 mRNA expression and protein content of pure dye fluoride group: With the exposure concentration, 24h and 48h in paragraph NaF can continue to induce osteoblast core transcription factor Nrf2 mRNA expression, but with exposure time was extended to 72h, this induction effect becomes obvious; cells exposed to 24h, 48h after NaF can continue to make the cytoplasm Nrf2 protein content decreased, while the cells after 48h exposure NaF can continue to make the nucleus Nrf2 protein content increased. tBHQ pretreatment group: tBHQ pretreatment plus fluoride treatment after 12h 24h, 48h and 72h, Nrf2 mRNA expression of anti-fluoride group compared with pure dye decline in the 24h segment nucleoprotein in Nrf2 protein content than the same group were transfected group increased fluoride . 7 of fluoride on osteoblasts HO-1 and NQO1 mRNA expression of pure dye fluoride group: pure dye fluorine 24h and 72h, rat osteoblasts HO-1 and NQO1 mRNA expression was not observed significant changes; single dye fluoro-48h , 0.25mmol / L group of intracellular HO-1 and NQO1 mRNA expression was significantly increased (P lt; 0.05); 0.50,2.00 and 4.00mmol / L group of intracellular HO-1 mRNA expression was significantly decreased compared with the control group (P lt; 0.05), and 4.00mmol / L group of intracellular NQO1 mRNA expression was significantly increased. tBHQ pretreatment group: 24h and 72h segment HO-1 mRNA expression of pure dye fluoro group decreased; in 48h paragraph, 4.00mmol / L group can induce HO-1 mRNA expression was increased; 24h segment 4.00mmol / L group intracellular NQO1mRNA pure expression of the same group of dye fluoro group decreased; 48h segment, 0.25 mmol / L group NQO1 mRNA expression compared with the same group of pure dye fluoro group decreased; 72h segment, 0.50,2.00,4.00 mmol / L group NQO1 mRNA expression compared to the same group The pure dye fluoro group decreased (P lt; 0.05). Conclusions 1 fluorine can inhibit the proliferation of osteoblasts, altering the cell cycle, but only the high dose (4.00mmol / L) only occur when osteoblast apoptosis significantly. 2 fluoride can occur in rat osteoblasts lipid peroxidation and DNA damage, but also can reduce the intracellular activity of antioxidant enzymes. 3 fluorine can induce osteoblast Nrf2 gene expression and protein content increased, thereby enabling Nrf2-ARE downstream target genes HO-1 and NQO1 expression, the cells antioxidant capacity enhancement. 4 In the present study conditions, tBHQ pre-osteoblast cells Nrf2 protein and HO-1 and NQO1 mRNA expression of pure dye fluoro group decreased.

The Effects of Fluoride on Nrf2-ARE Signal Pathway in Rat Osteoblasts

Abstract

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