Change and relationship between Gli1 and β-catenin on rats' bone formation with chronic fluorosis / 中华病理学杂志

To investigate the change and association of glioma-associated oncogene homolog 1 (Gli1) and β-catenin on bone formation in rats with chronic fluorosis which were inhibited by cyclopamine (Cycl). Forty-eight Sprague-Dawley rats were evenly divided to four groups, including control, F, F+Cycl and F+DMSO groups. The control group were fed with tap water (NaF1 ppm). The F, F+Cycl and F+DMSO groups were exposed to NaF (50 ppm) in drinking water as the chronic fluorosis model. Then the rats in F+Cycl or F+DMSO groups were injected by Cycl or DMSO after 6 months, respectively. Urine fluoride concentration was detected using fluorine ion selective electrode. The enzyme-linked immunosorbent assay (ELISA) was used to detect bone alkaline phosphatase (BALP). Bone tissues were stained with hematoxylin-eosin. The mRNA and protein expression of Gli1 and β-catenin in bone tissue were detected using real-time PCR, immunohistochemistry and Western blot. Compared with the controls, the urine fluoride concentration and the width and volume of bone trabeculae were increased in the F, F+Cycl and F+DMSO groups, but no statistical difference among the 3 fluorosis groups. The concentration of BALP was increased in the F group and decreased in F+Cycl group (0.05). The expression of Gli1 and β-catenin mRNA and protein was higher in the F and F+Cycl groups than controls, but lower in the F+Cycl group than in the F group. There was positive correlation between the expression of Gli1 and β-catenin (0.476, 0.05). The expression of Gli1 and β-catenin was also associated with BALP concentration and volume of bone trabeculae, respectively (0.457, (2)0.466, 0.581, 0.554, respectively, 0.05 for all). The expression of Gli1 can be inhibited by Cycl. It may be involved in the bone formation of rats with chronic fluorosis. It may also affect the expression of β-catenin, which is an osteogenesis factor.

Expression of calcineurin and nuclear factor of activated T cells 1 in testis of rats with chronic fluorosis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To discuss the significance of calcineurin (CaN) and nuclear factor of active T cells 1 (NFATc1) in the damage mechanism of the testis of rats with chronic fluorosis.</p><p><b>METHODS</b>Eighteen clear class SD male rats, aging 6 week-old, were randomly divided into 3 groups, 6 rats in each. The rats of control group were fed with tap water (NaF < 1 mg/L) and the experimental rats were exposed to NaF (lower group: 5 mg/L, higher group: 50 mg/L) to established the chronic fluorosis model. After 8 months, we observed the occurrence of dental fluorosis among rats in different groups, and the contents of urine fluoride were detected by fluorine ion selective electrode method. The body of the rats were weighted as well as their testis. The testis tissues were stained with hematoxylin-eosin and observed under light microscope to find the morphological changes. The expression of CaN and NFATc1's protein and mRNA in testis were detected by Immunocytochemistry (IHC) and In-situ hybridization (ISH).</p><p><b>RESULTS</b>The number of rats which was found dental fluorosis were separately 0, 4 and 5 in control group, low dose group and high dose group (χ(2) = 10.60, P < 0.05). The contents of urine fluoride were gradually increased in control group, low group and high group, which were (1.26 ± 0.17), (2.06 ± 0.64) and (7.69 ± 1.96)mg/L, respectively (F = 36.57, P < 0.05). The body weight were significantly different in all three groups(629.00 ± 16.00), (585.17 ± 17.27), (560.50 ± 16.07)g, F = 26.67, P < 0.05) and the testis weight were without statistical difference ((2.58 ± 0.17), (2.43 ± 0.31), (2.35 ± 0.38)g, F = 0.91, P > 0.05). Compared with the control group, the testicular structures were damaged in the experimental groups and especially significant in high dose group. The expression of CaN (59.10 ± 5.62, 77.93 ± 4.16, 101.69 ± 6.31, F = 74.18, P < 0.05) and NFATc1's (76.11 ± 4.41, 93.42 ± 3.85, 120.42 ± 9.31, F = 92.4, P < 0.05) protein in testis tissues were increased by the fluorine concentration. The mRNA expression of CaN and NFATc1 were separately (CaN: 58.76 ± 7.70, 82.01 ± 6.88, 99.47 ± 8.33, F = 42.65, P < 0.05 and NFATc1: 59.39 ± 4.74, 90.02 ± 5.37, 121.15 ± 7.69, F = 155.47, P < 0.05). There were positive correlation between the expression of CaN and NFATc1's protein and mRNA expression (r = 0.899, r = 0.908).</p><p><b>CONCLUSION</b>The changes in the signaling pathway of expression of CaN may be involved in the injury mechanism of testis tissues of rats with chronic fluorosis.</p>

Expression of mRNA and protein of p38, Osx, PI3K and Akt1 in rat bone with chronic fluorosis / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expressions of mRNA and protein of p38, Osx, PI3K, Akt1 in the rats bone with chronic fluorosis.</p><p><b>METHODS</b>Dental fluorosis were observed and the fluoride contents in the urine and bone were detected by fluorin-ion selective electrode. The morphologic changes and ultrastructure of rats' bone were observed by light and electronic microscopy. The expressions of protein and mRNA of p38, Osx, PI3K and Akt1 were detected by immunohistochemistry and real-time PCR, respectively. The contents of BALP and BGP in serum were detected by ELISA.</p><p><b>RESULTS</b>The rates of dental fluorosis in the fluorosis rats were increased, and the fluoride contents in bone and urine of the fluorosis rats were increased compared to the control group, the difference was statistically significant (P < 0.05). The bone trabeculae thickness and density and the thickness of bone cortex in fluorosis rats were remarkably increased, the space of bone trabeculae was reduced, and in accordance with the matching morphometrical indices, the difference was statistically significant (P < 0.05) as compared with the control rats. The contents of BALP [(54.61 ± 2.27) U/L] and BGP [(2.38 ± 0.16) µg/L]in the fluoride groups were higher than those in the control group, the difference was statistically significant (P < 0.05). Ultrastructurally, the broadening of the osseouslacuna was observed. The reduced protuberances of the osteocytes, the unclear organelle structure, pyknosis, karyotheca increasation and edged chromatin were also observed. Compared to the control group, the expressions of protein and its mRNA of p38, Osx, PI3K and Akt1 were higher in the fluorosis rats than those in the control rats, and the difference was statistically significant (P < 0.05). There is no any expression of p38, Osx, PI3K and Akt1 in the osteocytes in fluorosis rats.</p><p><b>CONCLUSIONS</b>The over-expression of p38, Osx, PI3K and Akt1 in bone tissue of fluorosis rats may relate to the accumulation of fluorine in the body. The bone injury mainly occur in the stage of the differentiation and proliferation. The upregulation of P38MARK signal path and PI3K/Akt1 signal path may be involved in the pathogenesis of bone injury caused by fluoride.</p>

Relationship of nuclear factor kappa B-related gene expression and osteoclast apoptosis induced by fluoride in bone tissue / 中国地方病学杂志

Objective To investigate the relationship between change of relevant gene of nuclear factor kappa B(NF-κB) and osteoclast apoptosis in bone injury of rats with chronic fluorosis,and to reveal the mechanism of skeletal fluorosis.Methods Thirty-six healthy SD rats,weighting 100-120 g,were randomly divided into three groups(twelve in each group).Rats of control group were fed with tap water(NaF ＜ 1 mg/L) and the experimental rats were exposed to NaF(low-dose group:5 mg/L,high-dose group:50 mg/L) through drinking water to established chronic fluorosis model.All rats were killed at the eight month and metaphysic of femoral was collected.Bone tissues were stained with hematoxylin-eosin and observed under optical microscope.Serum content of tartrateresistant acid phosphatase 5b (TRACP 5b) was detected by enzyme-linked immunosorbent assay (ELISA).Osteoclast was identified and counted by tartrate-resistant acid phosphatase staining(TRAP).The expression of p50,IκBα,Bcl-2 and Bax's mRNA and protein of bone tissue was detected by Real-time PCR and immunohistochemistry.Results Bone sclerosis was observed under optical microscope.The content of TRACP 5b in serum and the number of osteoclast in the low fluoride group[(3.45 ± 1.85)U/L,(6.75 ± 1.29)/slice]were significantly higher than that of the control[(1.26 ± 0.23)U/L,(3.92 ± 1.38)/slice,all P ＜ 0.05],but that of the high fluoride group [(2.74 ± 1.85)U/L,(3.33 ± 1.07)/slice]were lower than that of the low dose group(all P ＜ 0.05).The mRNA expressions of p50,IκBα,Bcl-2 and Bax in low fluoride group(4.41 ± 0.44,1.15 ± 0.25,2.02 ± 0.11,1.25 ± 0.22) were significantly higher than that of the control(1.46 ± 0.10,0.26 ± 0.07,1.00 ± 0.06,0.74 ± 0.09,all P ＜ 0.05),but the high fluoride groups' (0.69 ± 0.09,0.14 ± 0.03,0.95 ± 0.08,0.62 ± 0.08) were lower than that of the low dose group(all P ＜ 0.05).The protein expressions of p50 and IκBα in the low fluoride group (152.96 ± 7.87,156.20 ± 9.75) were significantly higher than that of the control(125.63 ± 9.85,118.97 ± 6.94,all P ＜ 0.05),but the high fluoride group(120.56 ± 9.57,114.50 ± 7.61) were lower than the low dose group(all P ＜ 0.05).The protein expressions of Bcl-2 and Bax(170.61 ± 6.60,160.77 ± 7.66) and the ratio of Bcl-2/Bax (1.07 ± 0.08) were higher than the control(l10.73 ± 5.27,114.64 ± 5.83,0.96 ± 0.04,all P＜ 0.05),but the high fluoride group(81.70 ± 8.00,99.93 ± 3.83,0.81 ± 0.08) were lower than that of the control and the low dose group (all P ＜ 0.05).There was a significant positive correlation between protein expression of p50,IκBα and Bcl-2/Bax (r =0.587,0.676,all P ＜ 0.05).Conclusions Chronic fluorosis can cause change of the relevant gene of NF-κB in rat bone tissues and osteoclast apoptosis.The mechanism of skeletal fluorosis might be related to the abnormal of osteclast apoptosis caused by changes of NF-κB p50 and IκBα.

Expression of nuclear factor kappa B-related mRNA and protein in bone tissue of fluorosis rats / 中国地方病学杂志

ObjectiveTo investigate the expression of nuclear factor kappa B(NF-kB)-related mRNA and protein in bone tissue of rats with chronic fluorosis.MethodsThirty-six healthy SD rats,weighting 100 - 120 g,were randomly divided into three groups (twelve in each group ).Rats of control group were fed with tap water (NaF ＜ 1 mg/L) and the experimental rats were exposed to NaF(low-dose group:5 mg/L,high-dose group:50mg/L) through drinking water.All rats were killed at the eight month and metaphysic of femoral was collected.Bone tissues were stained with hematoxylin-eosin and observed under optical microscope.Bone fluorine was detected by ashing-fluorin ion selective electrode method.Serum content of tartrate-resistant acid phosphatase 5b(TRACP 5b)was detected by enzyme-linked immunosorbent assay(ELISA).The expressions of p50,p65 and IkBα's mRNA and protein in bone tissue was detected by real-time PCR and immunohistochemistry.ResultsBone sclerosis was observed under optical microscope.The contents of bone fluorine in both the low and high doses fluoride groups [(6.32 ± 1.23),( 10.89 ± 1.56) mg/kg] were significantly higher than that of the control [(3.06 ± 1.01 ) mg/kg,all P ＜ 0.05],and of that the high fluoride group was significantly higher than that of the low fluoride group(P ＜ 0.05).Serum content of TRACP 5b of the low fluoride group[(3.45 ± 1.85)U/L] was significantly higher than that of the control[(1.26 ± 0.23)U/L,P ＜ 0.05],but that of the high fluoride group[(2.74 ± 1.85)U/L] was lower than that of the low dose group(P ＜ 0.05).The mRNA expressions of p50 and IkBα in the low fluoride group(4.41 ± 0.44,1.15 ± 0.25) were significantly higher than that of the control(1.46 ± 0.10,0.26 ± 0.07,all P ＜ 0.05),but that of the high fluoride group(0.69 ± 0.09,0.14 ± 0.03) was lower than that of the low dose group(all P ＜ 0.05).The protein expressions of p50 and IkBα in the low fluoride group(152.96 ± 7.87,156.20 ± 9.75) were significantly higher than that of the control( 125.63 ± 9.85,118.97 ± 6.94,all P ＜ 0.05),but the high fluoride groups' ( 120.56 ±9.57,114.50 ± 7.61 ) was significantly lower than that of the low dose group(all P ＜ 0.05).ConclusionFluoride can lead to altered gene expression of NF-kB pathway,and the latter may be involved in fluoride induced bone damage.

Effect of fluoride on expression of phosphoinositide 3-kinase, protein kinase B1 mRNA and protein in bone tissue of rats / 中国地方病学杂志

Objective To observe the expression of phosphoinositide 3-kinase(PI3K) and protein kinase B1 (Akt1) in PI3K/Akt signaling pathway in rat bones with fluorosis, and to reveal the mechanisms of the skeletal fluorosis. Methods Thirty-six SD rats were randomly divided into 3 groups (control group, low-dose fluorosis group, high-dose fluorosis group) and 12 rats were in each group according to body weight. The rats were fed with different concentrations of fluoride (NaF) to establish fluorosis models. Controls were fed with tap water( < 0.5 mg/L), experimental animals in low- or high-dose groups were fed with water containing NaF 5.0,50.0 mg/L, respectively. Rats were sacrificed after 6 months of treating with fluoride and the serum was kept for testing the bone metabolic markers of none gla protein(BGP) and cathepsin K(Cath-K) by enzyme-linked immunosorbent assay(ELISA), the proteins and mRNA levels of PI3K and Akt1 in rat bones were detected by immunohistochemistry and real time PCR, respectively. Results Each group of serum BGP and Cath-k were compared, the difference was statistically significant(F = 73.45,39.36, all P < 0.05). The contents of BGP[(1.99 ± 0.62), (2.38 ± 0.16)μg/L] and Cath-K [(89.07 ± 19.66), (110.16 ± 9.81)pmol/L] in the low-and high-dose fluorosis groups were higher than those in the control group[(0.15 ± 0.03)μg/L,( 18.32 ± 2.27)pmol/L], and the high fluorosis group was obviously higher than the low fluorosis group (all P < 0.05). Each group of serum PI3K and Akt1 protein and mRNA were compared, the difference was statistically significant(F- 178.16,118.08,38.81,52.31, all P< 0.05). Compared to the control group (181.55 ± 4.24,188.46 ± 2.18,3.84 ± 1.69,4.33 ± 0.89), the protein and mRNA expressions of PI3K(171.66 ± 2.85,154.12 ± 4.15,11.31 ± 4.18,20.54 ± 6.68), Akt1(177.47 ± 3.16,156.42 ± 3.18,12.52 ± 3.13,19.43 ± 5.36) were higher in the low- and high-dose fluorosis groups (all P < 0.05), and the high fluorosis group was obviously higher than the low fluorosis group (all P < 0.05). Conclusions BGP and Cath-K contents could be used as bone metabolic indices in the endemic fluorosis disease. Fluoride can increase the expression of PI3K and Akt1 mRNA and protein in bone tissue of fluorosis rats, and PI3K/Akt1 signaling pathway may be involved in the pathogenesis of bone injury caused by fluoride.