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Objective:To learn about the levels of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in bone tissue of rats with different types of skeletal fluorosis and analyze their correlation.Methods:Thirty 4-week-old SPF grade healthy SD rats were selected. After adaptive feeding for 1 week, the rats were divided into control group (4 ml·kg -1·bw deionized water + standard maintenance diet), osteosclerosis group [20 mg·kg -1·bw sodium fluoride (NaF) + standard maintenance diet], and osteoporosis/osteomalacia group (20 mg·kg -1·bw NaF + low-calcium and low-protein partial diet) according to their body weight (100 - 120 g) by random number table method, with 10 rats in each group, half male and half female; gavaged 6 days each week and the experimental period was 5 months. At the end of the experiment, samples of rat heart blood and lower limb femur were collected. The contents of serum methyl donor S-adenosylmethionine (SAM) and its metabolite S-adenosylhomocysteine (SAH) in serum, and the levels of 5-mC and 5-hmC in bone tissue were measured by enzyme-linked immunosorbent assay (ELISA). Western blot was used to determine the expression of DNA methyltransferase (DNMTs) and DNA hydroxymethylase (TETs) in bone tissue of rats. The correlation between serum SAM content, SAM/SAH ratio and bone tissue 5-mC level, and between the bone tissue 5-mC level and 5-hmC level was analyzed. Results:Serum SAM [11.03 (7.06, 18.63), 3.96 (2.32, 9.09), 3.91 (2.35, 4.46) nmol/L], SAH content [(4.69 ± 0.55), (5.41 ± 1.13), (13.90 ± 1.09) ng/L], SAM/SAH ratio [2.58 (1.54, 4.12), 0.62 (0.52, 1.69), 0.14 (0.13, 0.15)] and bone tissue 5-mC [103.39 (97.37, 109.35), 52.50 (50.19, 68.13), 55.03 (49.97, 59.57) ng/L], 5-hmC levels [(32.61 ± 8.84), (56.96 ± 8.48), (20.34 ± 6.22) ng/L] in the control group, osteosclerosis group and osteoporosis/osteomalacia group were compared, and the differences were statistically significant beween three groups ( H/ F = 12.81, 284.24, 21.85, 19.37, 55.23, P < 0.01). Compared with the control group, the content of SAM, the ratio of SAM/SAH, the level of 5-mC in the osteosclerosis group and osteoporosis/osteomalacia group, and the level of 5-hmC in the osteoporosis/osteomalacia group were lower ( P < 0.05), while the content of SAH in the osteoporosis/osteomalacia group and the level of 5-hmC in the osteosclerosis group were higher ( P < 0.05). Compared with the osteosclerosis group, the content of SAH in the osteoporosis/osteomalacia group was higher, while the ratio of SAM/SAH and the level of 5-hmC were lower ( P < 0.05). Western blot showed that there were statistically significant differences in the expression levels of DNMT1, DNMT3A, DNMT3B, TET1 and TET2 protein in bone tissue of rats in the control group, osteosclerosis group, and osteoporosis/osteomalacia group ( F = 285.45, 345.58, 239.83, 311.52, 318.24, P < 0.001). Among them, the expression levels of DNMT1, DNMT3A and DNMT3B protein in the osteosclerosis group and osteoporosis/osteomalacia group were lower than those in the control group, and the expression levels of DNMT1, DNMT3A and DNMT3B protein in the osteosclerosis/osteomalacia group were lower than those in the osteosclerosis group ( P < 0.05); the expression levels of TET1 and TET2 protein in osteosclerosis group were higher than those in the control group and osteoporosis/osteomalacia group, and the expression levels of TET1 and TET2 protein in the osteoporosis/osteomalacia group were lower than those in the control group ( P < 0.05). The results of Spearman rank correlation analysis showed that the content of SAM and the ratio of SAM/SAH in the control group, osteosclerosis group and osteoporosis/osteomalacia group were positively correlated with the level of 5-mC in bone tissue ( rs = 0.89, 0.92, 0.81, 0.73, 0.87, 0.73, P < 0.05). The levels of 5-mC and 5-hmC in bone tissue of rats in each group were negatively correlated ( rs = - 0.69, - 0.68, - 0.72, P < 0.05). Conclusions:The level of 5-mC in bone tissue of osteosclerotic fluorosis rats is low, and the level of 5-hmC is high, while those of osteoporosis/osteomalacia fluorosis rats are lower. The difference of 5-mC level in bone tissue of rats with different types of skeletal fluorosis is not significant, which may be related to the difference of 5-hmC level in bone tissue.
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Objective:To investigate the combined effect of fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation in rats.Methods:SD rats were divided into four groups by the method of random number table, namely normal nutrition group, low nutrition treatment group, fluoride exposure group and co-treatment of fluoride and low nutrition group according to 2 × 2 factorial experimental design, 8 rats in each group, half male and half female. Five months after the experiment, immunohistochemistry was used to test the expression levels of femoral alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL). Analysis of variance of factorial design was used to determine the interaction between fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation.Results:The immunohistochemical results of bone tissue showed that there were significant differences in the expression levels of osteogenesis differentiation markers ALP and Runx2 between different groups ( F = 25.98, 17.77, P < 0.001). Compared with normal nutrition group (0.005 2 ± 0.002 7, 0.003 1 ± 0.001 4), the expression levels of ALP and Runx2 in fluoride exposure group were higher (0.019 5 ± 0.005 0, 0.014 4 ± 0.004 4, P < 0.05). There was no significant difference between low nutrition treatment group (0.002 6 ± 0.001 8, 0.004 4 ± 0.003 2) and co-treatment of fluoride and low nutrition group (0.003 6 ± 0.000 7, 0.002 9 ± 0.000 8, P > 0.05). The expression levels of ALP and Runx2 in co-treatment of fluoride and low nutrition group were lower than those of fluoride exposure group ( P < 0.05). There were significant differences in the expression level osteoclastic differentiation marker of RANKL and the ratio of RANKL/OPG ( F = 10.50, 31.05, P < 0.001). Among them, the RANKL/OPG ratio (0.115 3 ± 0.039 5) in fluoride exposure group was lower than that in normal nutrition group (1.426 3 ± 0.777 2), and the RANKL expression level and RANKL/OPG ratio (0.019 5 ± 0.007 7, 7.258 7 ± 3.674 3) in co-treatment of fluoride and low nutrition group were higher than those in normal nutrition group (0.004 4 ± 0.002 5, 1.426 3 ± 0.777 2, P < 0.05). However, there was no significant difference in the RANKL expression level and RANKL/OPG ratio (0.004 0 ± 0.001 9, 2.022 3 ± 0.753 7) in low nutrition treatment group ( P > 0.05). The expression level of RANKL and the ratio of RANKL/OPG in the co-treatment of fluoride and low nutrition group were higher than those in low nutrition treatment group and fluoride exposure group ( P < 0.05). The 2 × 2 analysis of variance of factorial design showed that fluoride exposure and low nutrition had interaction on ALP, Runx2, RANKL expression levels and RANKL/OPG ratio ( F = 4.38, 19.39, 22.12, 108.00, P < 0.05), antagonistic effect on ALP and Runx2 expression, synergistic effect on RANKL expression and RANKL/OPG ratio. Conclusions:In rat bone tissue, fluoride exposure promotes osteogenesis differentiation, inhibits osteoclastic differentiation dominated by active osteogenic function. The interaction between fluoride and low nutrition on osteogenesis and osteoclastic differentiation is antagonistic osteogenesis differentiation and synergistic promotion of osteoclastic differentiation. Normal nutrition conditions are material basis of osteogenesis differentiation, and low nutrition is the inducement of enhanced osteoclastic differentiation.
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Objective:To establish a protein-protein interaction (PPI) network of 5 microRNA (miRNA) target genes differentially expressed in the plasma of coal-burning fluoride exposed population, and to screen genes and gene modules with important roles.Methods:Five miRNA (hsa-miRNA-3131, hsa-miRNA-4516, hsa-miRNA-6501-5p, hsa-miRNA-10b-5p, hsa-miRNA-4683) target genes differentially expressed in the plasma of coal-burning fluoride exposed population screened by our previous study were mapped to the STRING online database (https://string-db.org), and the PPI network was screened. The Cytoscape v3.6.0 software was used to visualize the PPI network, the topological attribute values degree and betweenness centrality were obtained by the NetworkAnalyzer plug-in, and the central node was filtered in the network (the node with the highest degree and the highest betweenness centrality). At the same time, the maximal clique centrality (MCC) analysis method in the CytoHubba plug-in was used to determine the important nodes in the PPI network. The cluster analysis was conducted by the MCODE plug-in, and the gene modules were screened in the PPI network. The protein names contained in the gene modules were submitted online to the KOBAS v3.0 database (http://kobas.cbi.pku.edu.cn/), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the gene modules selected by the MCODE plug-in.Results:The PPI network of target genes was consisted of 1 035 nodes and 4 346 edges. The degree (101) and betweenness centrality (0.010 723 89) of ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA52) were the highest, which was the central node of the PPI network. According to MCC analysis, UBA52 was an important node in the PPI network. The top 5 gene modules were selected from the PPI network, and the highly enriched gene pathways in the KEGG pathway enrichment analysis of the 5 gene modules included ubiquitin mediated proteolysis, spliceosome, endocytosis, neuroactive ligand-receptor interaction and vesicular transport.Conclusion:The PPI network of 5 miRNA target genes differentially expressed in the plasma of people exposed to coal-burning pollution of fluoride is successfully established, and the UBA52 gene and the 5 main pathways of gene modules are selected.
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Objective To observe the efficacy and feasibility of transplanting autologous bone marrow mesenchymal stem cells (BMSCs) via articular cavity on skeletal fluorosis rabbits. Methods A total of 30 rabbits, half male and half female, were divided into control group (n=6) and experimental group (n=24) by random number table method. The two groups of rabbits were given drinking water containing NaF 0 or 300 mg/L, respectively. After 90 days, 24 rabbits were divided into single treatment group, multiple treatment group, spontaneous recovery group and treatment solvent control group (6 rabbits in each group, half male and half female). After isolation, the BMSCs from skeletal fluorosis rabbits were cultured and identified, autologous BMSCs were transplanted into rabbit bodies via articular cavity at once or at three different other times, respectively. After 60 days, femur image was observed through X-ray. Femur bone mineral density was measured with quantitative CT (QCT). Serum alkaline phosphatase (ALP) and osteocalcin (BGP) were also measured using enzyme linked immunosorbent assay (ELISA). Bone fluoride content was determined via the fluorine ion-selective electrode method. Histopathology changes of femur were observed through HE staining and the trabecular area was calculated. Results In the multiple treatment group, patchy high-density images of femur were disappeared and abnormal bone texture was recovered compared with that of before transplantation. Bone density [(536.91 ± 25.51) g/cm3], ALP concentration [(20.06 ± 6.25) U/L], BGP concentration [(1230.01 ± 119.50) μg/L], bone fluoride content [(1442.40 ± 458.54) mg/kg] and trabecular area [(27.81 ± 2.90) Tb.Ar] of the multiple treatment group were lower than those of spontaneous recovery group [(635.11 ± 93.42) g/cm3, (43.08 ± 2.82) U/L, (3207.73 ± 788.80) μg/L, (2557.65 ± 173.90) mg/kg, (38.52 ± 2.81) Tb.Ar], and th e differences were statistically significant (P<0.05). HE staining in the multiple treatment group showed that bone marrow cavity was enlarged, and the number of trabeculae was decreased, accompanied by some new generated, neatly arranged normal trabeculae. But similar results were not observed in the single treatment group. Conclusion After multiple transplantation via articular cavity, autologous BMSCs from skeletal fluorosis rabbits could repair the damaged bone tissue and improve the pathological damage of skeletal fluorosis with osteosclerosis.
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Objective To establish an model of fluorosis with human primary osteoblasts in vitro and to detect the influences of different doses of sodium fluoride (NaF) on histone acetylation of CyclinD1,cyclindependent kinases 4 (CDK4) gene in human osteoblasts,then to explore the molecular mechanism of skeletal fluorosis from epigenetic perspective of the cell cycle regulation related genes.Methods Human primary osteoblasts from bone tissues of trauma surgery healthy people (car accident) were isolated by enzyme digestion and identified.The osteoblasts were treated with 0,125,250,500 and 1 000 μmol/L NaF for 72 h.The level of histone acetylation (H3K9,H3K14,H4K12,H4K16) in the transcription regulatory region (ChIP1 region) and in the coding region (ChIP2 region) of CyclinD1 and CDK4 genes were detected by quantitative chmmatin immuno-precipitation (Q-ChIP).Results ①After human osteoblasts were treated with 0,125,250,500 and 1 000 μmol/L NaF,respectively,the levels of histone acetylation of H3K9 in ChIP1 transcription regulatory region of CyclinD1 gene were 1.152 ± 0.104,1.174 ± 0.187,1.090 ± 0.176,1.170 ± 0.197 and 1.147 ± 0.097,respectively,the differences were not statistically significant (F =0.524,P ＞ 0.05);the average levels of histone acetylation of H3K14 were 1.495 ± 0.117,1.465 ± 0.069,1.470 ± 0.187,1.760 ± 1.089 and 1.341 ± 0.443,the differences were not statistically significant (F =0.841,P ＞ 0.05);the levels of histone acetylation of H4K12 were 1.239 ± 0.286,0.702 ± 0.063,0.765 ± 0.370,1.011 ± 0.321 and 1.319 ± 0.026,the differences were not statistically significant (F =2.329,P ＞ 0.05);the levels of histone acetylation of H4K16 were 1.452 ± 0.217,1.621 ± 0.165,1.462 ±0.090,1.510 ± 0.146 and 1.564 ± 0.154,the differences were not statistically significant (F =0.123,P ＞ 0.05).②The levels of histone acetylation of H3K9 in ChIP1 transcription regulatory region of CDK4 were 1.472 ± 0.163,1.580 ± 0.161,1.585 ± 0.132,1.451 ± 0.136 and 1.560 ± 0.039,the differences were not statistically significant (F =0.461,P ＞ 0.05);the levels of histone acetylation of H3K14 were 0.919 ± 0.149,0.900 ± 0.059,0.911 ±0.162,0.663 ± 0.049 and 0.841 ± 0.122,the differences were not statistically significant (F =0.974,P ＞ 0.05);the levels of histone acetylation of H4K12 were 0.456 ± 0.142,0.911 ± 0.126,0.969 ± 0.185,1.110 ± 0.146 and 0.931 ± 0.141,the differences were not statistically significant (F=5.459,P ＞ 0.05);the levels of histone acetylation of H4K16 were 1.315 ± 0.083,1.374 ± 0.153,1.423 ± 0.055,1.300 ± 0.132 and 1.385 ± 0.696,the differences were not statistically significant (F =1.663,P ＞ 0.05).③The differences of histone acetylation levels of H3K9,H3K14,H4K12 and H4K16 in ChIP2 coding region of CyclinD1 and CDK4 genes were not statistically significant between NaF treatment groups (F =0.392,0.823,0.999,0.397,0.705,0.049,1.065,0.196,P ＞ 0.05).Conclusion The histone acetylation of CyclinD1 and CDK4 may not be involved in the transcriptional regulation in human primary osteoblasts treated with fluoride.
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Objective To detect the early diversification of the bone mineral density in skeletal fluorosis of rabbits by quantitative computed tomography (QCT),and analyze the possible relationship between bone mineral density and bone injury in rabbits with fluorosis.Methods A total of 16 rabbits,half male and half female,were randomly divided into control and experimental groups according to body weight.The two groups of rabbits were given drinking water containing NaF 0 or 300 mg/L,respectively,for 90 days.After the experiment,their bone fluoride content was determined via the fluorine ion-selective electrode method.The alkaline phosphatase (ALP) and osteocalcin (BGP) in serum were measured using enzyme-linked immunosorbent assay (ELISA).Femurl bone mineral density was detected with QCT in vivo.Histopathological changes of femur were observed under light microscope and trabecular acreage was calculated.The results were analyzed with independent-samples t test(t') and partial correlations.Results The bone fluoride content [(3 232.16 ± 927.85) mg/kg],ALP [(42.69 ± 3.28) U/L],BGP concentration [(2 504.19 ± 276.79) μg/L],bone density [(653.49 ± 167.81) g/cm3] and trabecular number [(39.02 ± 3.33)Tb.Ar] of the experimental group were higher than those of control group [(554.01 ± 376.51)mg/kg,(20.50 ± 4.90)U/L,(1 294.60 ± 191.86)μg/L,(540.40 ± 41.99)g/cm3,(8.15 ± 2.34)Tb.Ar],and the differences were statistically significant (t'=7.565,10.641,10.158,2.615,14.494,all P ＜ 0.05).The tissue sclerosis,bone sclerosis and bone texture coarsening were observed through bone mineral density imaging taken by QCT in experimental group.The number of trabeculae increased and the arrangement of tra bec ulae was disorganized.Bone mineral density was positively correlated with bone fluoride,trabeculae,BGP and ALP (r =0.702,0.627,0.614,0.567,all P ＜ 0.05).Conclusions QCT bone density measurement in skeletal fluorosis of rabbits can be used to compute the threedimensional bone density.And it has a good correlation with bone fluoride content,bone histopathological changes and index of bone metabolism in skeletal fluorosis,which suggests that QCT may provide a useful reference for application in patients with skeletal fluorosis.
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Objective To establish an in vitro model of primnary osteoblasts fluorosis and to detect the influences of different doses of fluorosis on promoter methylation,transcription and expression of p16,then study the epigenetic effects of p16 gene on skeletal fluorosis.Methods Osteoblasts were isolated from Sprague-Dawley neonatal rats by enzyme digestion,and identified by morphology,alkaline phosphatasc staining and alizarin red staining.Osteoblast were treated with 0 (control group),200,400,800 and 1 600 μmol/L NaF for 72 h.The pl6 gene promoter region was amplified in the transcription initiation site-88-+ 143 region by bisulfatesequencing polymerase chain reaction (BSP).The mRNA transcription and the protein expression of p16 were detected by real-time quantitative PCR and Western blotting.Results Among the groups of osteohlasts treated with 200,400,800,1 600 μmol/L NaF,the positive rates of DNA methylation of promoter region in p16 gene of osteoblasts were 5.88％ (10/170),12.94％ (22/170),17.65％ (30/170) and 33.53％ (57/170),respectively.No DNA methylation was observed in the control group.There were significant differences between the control group and the NaF-treated osteoblasts groups (x2 =92.87,P ＜ 0.05).Average levels of p16 mRNA were 1.050 ± 0.073,0.869 ±0.037,1.065 ± 0.118 and 0.786 ± 0.148 in 200,400,800 and 1 600 μmol/L NaF-treated osteoblasts groups,compared with the control group (1.110 ± 0.315),there were significant differences among groups (all P ＜ 0.05) and 1 600 μmol/L NaF-treated osteoblasts group was much lower than other groups (P ＜ 0.05).Average levels of p16protein were 1.190 ± 0.050,1.214 ± 0.058,1.122 ± 0.123 and 0.320 ± 0.074 in 200,400,800 and 1 600 μmol/LNaF-treated osteoblasts groups,compared with the control group (1.115 ± 0.057),there were significant differences among groups (all P ＜ 0.05) and 1 600 μmol/L group was much lower than other groups.Conclusion NaF can cause hypermethylation in the promoter region of p16 gene,then suppress the expression of mRNA and protein,which might be one of the important mechanisms of cell proliferation change and cell cycle disorder in skeletal fluorosis.
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0.05),but the average levels of DNMT1 mRNA of mild and moderate groups were significantly lower than that of the control group(P