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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.
ABSTRACT
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 investigate the role of anaphylatoxin C3a on epithelial mesenchymal transition(MTT) in normal human bronchial epithelium cells and its molecular mechanism.Methods Normal human bronchial epithelium cells BEAS-2B were cultured,and divided into control group,rhC3a stimulation group,rhC3a+ C3a receptor(C3aRA)antagonist group,the morphological changes of cells were observed by microscope;cell proliferation was detected by MTT;the expression of TGF-β1 protein level in cell supernatant was evaluated by ELISA;the expression of C3aR mRNA and EMT related indicators mRNA changes were detected by RT-PCR;the expression of C3aR and Smad2/3,p38 MAPK pathway proteins were detected by Western blot.Results Cell morphology in 30,50 nmol/L rhC3a stimulation group was changed from normal cobblestone like to spindle shape,cell morphology in C3aRA antagonist group had no significant change when compared with the control group.The cell proliferation was reduced in 50 nmol/L rhC3a stimulation group(P=0.047);the levels of TGF-β1,p-Smad2/3,p-p38-MAPK protein were increased(P＜0.05),C3aR mRNA and protein levels were also significantly increased (P＜0.05) in 30 nmol/L rhC3a stimulation group when compared with control group,but the addition of 1 μmol/L C3aRA could reduce their expressions(P＜0.05).30 nmol/L rhC3a could induce the up regulation of α-SMA and N-cadherin mRNA,and decrease the expression of E-cadherin mRNA,adding 1 μmol/L C3aRA can antagonize this process (P＜0.05).Conclusion Anaphylatoxin C3a can induce EMT in normal human bronchial epithelium cells by combining C3aR,its mechanism may be involved in activating Smad2/3 and p38-MAPK pathway.