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BACKGROUND: Long-term excessive intake of fluoride, especially through drinking water, can cause chronic fluorosis of bone. The disease can lead to bone damage and deformity, and is difficult to recover.Unfortunately, we have not developed a noninvasive or minimally invasive method for its early diagnosis. OBJECTIVE: To observe the expression of apoptosis-related miRNAs under the action of excessive fluorine in human osteoblasts. METHODS: The fluorine model was established in the human osteoblasts by cultured with 20 and 40 mg/L sodium fluoride for 24 and 48 hours, respectively. The expression levels of apoptosis-related miRNAs were determined by PCR array. RESULTS AND CONCLUSION: After 24-hour treatment of sodium fluoride, 48 kinds of miRNAs were upregulated and 4 ones were down-regulated in the osteoblasts. After 48-hour treatment of sodium fluoride, 21 kinds of miRNAs were upregulated and 2 ones were down-regulated. It showed that nine up-regulated miRNAs and one down-regulated miRNA were same in two periods. The 10 miRNAs are selected for target gene analysis on bioinformatics software that refer to the effect of anti-apoptosis and pro-apoptosis, which is of great significance for the early identification of skeletal fluorosis.
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ObjectiveTo study the relationship between expression of a3 chain of collagen Ⅸ (COLIXA3)mRNA in the population exposed to fluorine and fluorosis,in order to reveal the role of COLIXA3 gene in the pathogenesis of endemic fluorosis.MethodsTwelve cases of mild drinking water-born skeletal fluorosis were selected as case groups in Regiment 123 and 128 of Xinjiang Production and Construction Corps Seven Division,6cases of healthy people living in fluorosis areas for more than 10 years as a internal control group and 6 heathly cases living in non-fluorosis areas for more than 10 years as a external control group.The expression of COLIXA3mRNA of peripheral blood lymphocyte of skeletal fluorosis patients and control groups were determined by using SYBR Green Ⅰ chimeric fluorescent method for real-time quantitative PCR.ResultsThe results of the relative expression of COLIXA3 mRNA of case group,internal control group and external control group were 2.16 ± 0.62,1.06 ± 0.09 and 1.05 ± 0.12,respectively.The COLIXA3 expression in case group was significantly higher than that of the internal control group and the external control group (all P ＜ 0.05),while the difference of COLIXA3expression between the internal control group and the external control group was not significantly different (P ＞0.05).ConclusionsFluorine contributes to the expression of COLIXA3 mRNA in peripheral blood lymphocyte,and the expression is up to 2 times higher than that of the control groups,meaning potential biomarkers.
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Objective To study the COLIXA3 gene polymorphism of patients with fluorosis and to explore the pathogenesis of COLIXA3 gene in endemic fluorosis.Methods Fifty one cases of patients with drinking-water borne fluorosis were selected as the case group in Xinzhou city,Shanxi province and 28 cases of healthy people were as the control group.Dental fluorosis was detected by Dean method and skeletal fluorosis was examined by X-ray.COLIXA3 of exon 5 gene product of 103 points was amplified by PCR and the gene locus genotype was sequenced.Results Ten cases of mild dental fluorosis,14 cases of moderate dental fluorosis,15 cases of severe dental fluorosis were detected among the 51 patients.The control group was free of dental fluorosis.All the 51 cases of patients with fluorosis had varying degrees of skeletal fluorosis,mainly osteosclerosis lesions,accounting for 86.27％(44/51 ),and mild skeletal fluorosis patients were all osteosclerosis lesions,and osteosclerosis lesions and multiple skeletal lesions were found among moderate and severe skeletal fluorosis patients in the case group,while control group had no skeletal fluorosis.The differences between genotypes of frequency distribution of AA,Aa,aa of COLIXA3 of case and control groups were not statistically significant [96.08％(49/51 ),3.92％(2/51 ),0.00％(0/51) and 96.43％(27/28),3.57％(1/28),0.00％(0/28),x2 =0.94,P ＞ 0.05].ConclusionsCOLIXA3 gene polymorphism is not significantly correlated to fluorosis.
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Objective To study the effect of fluoride on expression of osteoblast Runx2, Osterix and their downstream COL I A2 in vitro. Methods Human osteoblast Saos-2 was cultured in vitro. The cells were grouped according to fluoride(NaF) dose used: 0(control ), 0.625,1.250,2.500,5.000,10.000,20.000,40.000,80.000,160.000 mg/L. Cells were collected after 24 h culture, RNA extracted, and the mRNA expression of Runx2 and Osterix and downstream genes COL I A2 was detected using fluorescent quantitative reverse transcription polymerase chain reaction [Real-time (RT)-PCR]). Results After 24 h in vitro cell cultivation with NaF, the expression of Runx2 in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups(388.00 ± 41.80,209.00 ± 25.80,42.80 ±4.52,63.00 ± 16.10,24.30 ± 4.23,16.20 ± 4.32) was higher than that of the control group( 1.00 ± 0.12, all P ＜0.05). The expression of Runx2 in 40.000,80.000,160.000 mg/L groups(0.40 ± 0.05,1.91 ± 0.28,4.87±1.36)compared with that of control group, the difference was statistically insignificant(all P ＞ 0.05).The expression of Osterix mRNA in 1.250,2.500,5.000 mg/L groups(4.04 ± 1.67,229.00 ± 51.00,46.40 ± 10.60) was higher than that of the control group( 1.00 ± 0.42,all P ＜ 0.05). The expression of Osterix mRNA in 10.000,20.000,40.000,80.000,160.000 mg/L groups(0. 16 ± 0.07,0.13 ± 0.01,1.73 ± 0.54,0.01 ± 0.01, 0.09 ± 0.01) compared with that of control group, the difference was statistically insignificant (all P ＞ 0.05). The expression of COL I A2 mRNA in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups (2.27 ± 0.89,8.03 ± 2.31,14.20 ± 2.75,7.66 ± 1.34,8.96 ±2.30) was higher than that of the control group (1.00 ± 0.04, all P ＜ 0.05). The expression of COL I A2 mRNA in 160.000 mg/L(0.54 ± 0.01 ) was lower than that of the control group(P ＜ 0.05). Conclusions Fluoride may affect mRNA expression of Osterix and Runx2 in osteoblast and their expression level is related to fluoride concentration.Runx2 and Osterix can also regulate the expression of COL I A2 mRNA.
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Objective To observe the lecithin's effect on membrane of African green monkey kidney cells (Vero) exposed to sodium arsenite(NaAsO2). Methods Vero cells cultured in vitro were divided into 4 groups:control group (saline), model group (2.20 mg/L NaAsO2), high eoncentration of lecithin and arsenic group (53.33mg/L lecithin + 2.20 mg/L NaAsO2), low eoncentration of lecithin and arsenic group( 13.32 mg/L lecithin + 2.20 mg/L NaAsO2), 6 bottles of cells in each group, medium was changed every 2 days, cultured for 120 h. Na+ ,K+-ATPase activities of membrane were measured by spectrophotometry, and membrane phospholipids composition including phosphatidylserine (PS), phosphatidylethano-lamine (PE), phosphatidylcholine (PC) and sphingmyelin (SM) were measured by high performance liquid chromatography (HPLC). Results The Na~, K+-ATPase activities of membrane of control group, model group, high concentration of lecithin and arsenic group, low concentration of lecithin and arsenic group were (0.962 ± 0.081) × 106, (0.544 ± 0.037) × 106, (0.647 ± 0.043) x 106, (0.550±Compared with control group, the Na+ ,K+-ATPase activities of other 3 groups were significantly reduced (all P 0.05). Compared with control group[(0.087 ± 0.003), (0.127 ± 0.053), (0.588 ± 0.105),(0.071 ± 0.029)g/L], PS, PE, PC, SM levels in model group[(0.051 ± 0.018), (0.073 + 0.030), (0.240 ±0.038), (0.047 ± 0.121 )g/L] were significantly lower(all P 0.05), but SM[(0.057 ± 0.004)g/L] significantly decreased(P 0.05]. Compared with model group,the levels of PS, PE, PC, SM in high concentration of lecithin and arsenic group were significantly higher(all P 0.05), and PC was significantly higher(P < 0.05). Conclusions High concentration lecithin has certain protective effect on Vero cell membrane exposured to sodium arsenite.
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Objective To explore whether different degrees of fluorosis influence the expression of cartilage COLIXA3 protein in fluorosis model rats. Methods Forty male Wistar rats 3 to 4 weeks old were randomly divided into 5 groups according to body mass, and these rats were fed with distilled water containing sodium fluoride(NaF) of 0(control), 25, 50, 100 and 150 mg/L for 6 months, respectively, in order to establish the animal model of drinking water type fluorosis. Pathomorphologieal changes of the osseous tissues of rats were analyzed under light microscope and transmission electron microscope, and the expression of COLIXA3 protein of femur metaphysis was examined by immunohistochemistry. Results HE staining showed different degrees of femoral metaphyseal ossification of cartilage in each experimental group, bone density increased, with sclerotic lesions of skeletal fluorosis. The control group showed no abnormal cartilage. Electron microscopy showed that the experimental groups with varying degrees of cartilage cell swelling, cell matrix fades, 50 mg/L group .showed hyperplasia, and 100,150 mg/L groups were observed with organelles decreased, part of the disintegration of the cartilage cell lacunae, lmmunohistochemical staining of rat chondrocytes COLIXA3 was positive, cytoplasm with brown granules, cartilage COLIXA3 protein expression(23.3 ± 4.5, 41.2 ± 5.6, 26.4 ~ 7.5) in the 25, 50 and 100 mg/L groups enhanced. Compared to the control group (6.1 ± 3.5), the expression of 50 and 100 mg/L groups was significantly increased, and the differences were statistically significant(all P 0.05). Conclusions There has pathological changes of sclerosing skeletal fluorosis in animal model. Low-dose fluoride promotes while high-dose inhibits cartilage cell proliferation. When fluorine concentration in external environment is too high and with extended exposure to fluoride, direct toxic effects of fluoride on cartilage cells is observed. Fluorine affects and promotes the expression of COLIXA3 protein in cartilage. Low-dose fluoride can promote COLIXA3 protein expression, as the dose increases (over 100 mg/L), the effect decreases.
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Objective To study the effects of fluoride on minichromosone maintenance(MCM)3 mRNA and the bone formation-related gene:bone sialoprotein(BSP),osteocalcin(OC),osteopontin(OP)mRNA expression on human osteoblast cells.The expression of MCM3 was tested for diagnosis and surveillance value on osteoblast treated with excess fluoride.Methods Human osteoblast cell(Saos-2)was cultured in McCoy5A medium and treated with fluoride(sodium fluoride,NaF).There were eight groups including:0(control),0.625,1.250,2.500,5.000,10.000,20.000,40.000 mg/L groups.Expression of MCM3,BSP,OC,OP mRNA were detected by real-time PCR.Dual-standard curve method was used for analysis.ALPase was determined by measuring the absorbance using a micro titer plate reader. Results Expression of MCM3 mRNA was lower in the 0.625,1.250,2.500,5.000,20.000, 40.000 mg/L groups(0.059 ± 0.003,0.027 ± 0.001,0.272 ± 0.004,0.115 ± 0.002,0.137 ± 0.004,0.754 ±0.002, all P > 0.05) and was higher in10.000 mg/L group(21.300 ± 1.200, P < 0.01 ) than control group( 1.000 ±0.020), especially 10.000 mg/L group was higher than groups treated with fluoride(all P < 0.01 ), the differences among groups were significant(F = 305.842, P < 0.01 ). Expression of BSP mRNA was significantly higher in 0.625,1.250,2.500,5.000,10.000 mg/L groups(71.80 ± 3.60,133.00 ± 7.20,85.50 ± 0.60,80.90 ± 1.20,304.00 ± 21.00)than the control group( 1.00 ± 0.04), especially 10.000 mg/L group was higher than others groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 159.531, P < 0.01 ). Expressions of OC mRNA were higher in 0.625,1.250,2.500,5.000 mg/L groups(110.00 ± 12.00,143.00 ± 2.10,90.60 ± 4.10,23.70±1.20) than control group(1.00 ± 0.01, all P < 0.01), and the differences among groups were significant (F = 158.734, P < 0.01 ). Expression of OP mRNA were higher in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups(167.00 ± 11.20, 111.00 ± 12.10,72.50 ± 3.50,134.00 ± 14.00,42.30 ± 2.40,45.20 ± 3.30) than the control group(1.00 ± 0.04, all P < 0.05 or < 0.01 ), the differences among groups were significant(F = 60.226, P < 0.01 ).Compared with control group(4.2 ± 1.2), the ALPase activity was increased in all groups treated with fluoride (6.0 ± 0.4,5.8 ± 0.1,5.7 ± 0.4,7.7 ± 1.1,19.2 ± 2.4,8.5 ± 3.0,18.1 ± 4.2), but only 10.000 mg/L and 40.000 mg/L groups were higher than control group and other groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 7.806, P < 0.01 ). Conclusions Irregular expression of MCM3 mRNA is not suitable as a diagnostic and monitoring biomarker of osteoblasts exposed to excessive fluoride. Fluoride may affect the osteoblast-related gene expression and to promote osteogenic differentiation.
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<p><b>OBJECTIVE</b>Cigarette smoke extract (CSE) can induce injuries of pulmonary II epithelial cells, activate nuclear factor-kappaB and increase tumor necrosis factor-alpha(TNF-alpha) secretion. This study aimed to investigate whether azithromycin can protect pulmonary II epithelial cells from injuries induced by CSE and relevant mechanisms.</p><p><b>METHODS</b>Pulmonary II epithelial cells (A549 cells) were cultured in vitro. After 48 hrs of culture the cells were randomly treated with serum-free DMEM only (blank control group), azithromycin + serum-free DMEM, CSE+ serum-free DMEM or CSE+azithromycin. Eight hours later the morphology of A549 cells, the activity of NF-kappaB and the levels of TNF-alpha were measured by inverted microscope, immunohistochemistry and ELISA.</p><p><b>RESULTS</b>The morphology and structure of A549 cells were changed, NF-kappaB activity increased (dark brown staining ) and TNF-alpha levels (0.307 +/- 0.036 pg/mL vs 0.234 +/- 0.028 pg/mL)increased in the CSE+ serum-free DMEM group compared with the blank control group (P < 0.01). CSE together with azithromycin treatment recovered partly the morphological injuries of A549 cells. It also attenuated NF-kappaB staining and decreased TNF-alpha levels from 0.307 +/- 0.036 pg/mL (CSE+serum-free DMEM group) to 0.269 +/- 0.009 pg/mL (P < 0.05).</p><p><b>CONCLUSIONS</b>Azithromycin may inhibit NF-kappaB activity, decrease TNF-alpha secretion and thus lessen cytotoxicity of CSE to A549 cells.</p>