| Objective: In this study, we explored expression of factors related to the Hedgehog(Hh) signaling pathway in the rats treated with excessive fluorine in vivo and in vitro, and investigated the effects of silencing the Smo gene on expression of these factors and apoptosis of rat primary chondrocytes in vitro. Our aims were to explore the pathogenesis of fluorosis and to provide an experimental basis for the prevention and treatment of fluorosis. Methods:(1) In vivo studies: Thirty-six healthy Sprague-Dawley(SD) rats were randomly divided into three groups(6 males and 6 females per group), i.e. a control group(fed with a drunking water containing Na F <1 mg/L) and two fluorosis groups(fed with a drunking water containing Na F of 5 mg/L and 50 mg/L, respectively). After 6 months of experiment treatment, the rats were were euthanized using bloodletting in the femoral artery, the contents of fluorine urine and fluorine bone were detected with the fluorin-ion electrode method. Histological changes in cartilage tissues were observed on Hematoxylin & Eosin(H&E) stained under light microscope. Protein level of components in the Hh signaling pathway, including Shh, Smo and BMP-2, as well as the apoptosis-regulating proteins Bcl-2 and Bax, were detected using immunohistochemistry staining(IHC) and Western blotting.(2) In vitro studies: Primary cartilage cells were obtained from articular cartilage of 4 neonatal Sprague-Dawley rats using a mechanical-enzymatic digestion method. The primary chondrocytes were identified with toluidine blue. Logarithmic phase cells were divided into groups based on their fluorine concentration [Groups had fluorine concentrations of 0(control), 0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 m M, respectively]. After cultured for 72 h, the cell viability was determined using MTT assay, which provided data for determination of an optimal fluorine concentration. Smo si RNA1~3 were transfected into chondrocytes by lentivirus vector. After 72 h of infection, the Smo level was detected using Western blotting for selection of the best Smo si RNA. The experiment was divided into blank control group, fluorosis group, control si RNA group and Smo si RNA group. After cultured for 72 h, expressions of Shh, Smo and BMP-2 were detected using Western blotting and RT-PCR, while expressions of Bcl-2 and Bax were detected using Western blotting, and the apoptosis of chondrocytes was assessed using a flow cytometry. Results:(1) The fluorine contents in the urine and bone in low and high-dose fluoride groups(3.66±0.43, 402.38±33.77; 8.05±0.60, 935.12±49.60) were higher than control group(1.12±0.16, 106.45±11.28)(P<0.05).(2) Observations under a light microscope showed that in the control group the cartilage edge was tidy and cells were organized in a column. As the amount of stained fluorine rose, there were different degrees of cartilage stem epiphyseal ossification, and the bone trabecula were widened.(3) The protein levels of Shh, Smo, BMP-2 and Bax in the fluorosis groups(0.86±0.09, 0.92±0.11, 1.02±0.10, 0.91±0.14; 1.11±0.15, 1.17±0.15, 1.13±0.12, 0.92±0.11) were significantly increased than in the control group(0.62±0.07, 0.74±0.09, 0.77±0.11, 0.63±0.06), while Bcl-2 was significantly decreased in the fluorosis groups(0.78±0.03; 0.57±0.09) than in the control group(0.84±0.10)(P<0.05).(4) In the primary chondrocytes cytoplasm was appear purple metachromatic granules, this suggests that the cartilage cells was extracted succeed.(5) Compared with the control group, cell vitality was increased in the groups treated with 0.125, 0.25 and 0.5 m M fluoride(120.83±7.21, 123.17±4.62 and 118.51±10.09, respectively) and decreased in the groups treated with 1.0, 2.0, 4.0 m M fluoride(82.13±6.25, 35.16±4.84 and 7.03±2.23, respectively)(P<0.05).(6) The Smo si RNA1~3 could decrease the Smo protein level in the chondrocytes, with Smo si RNA2 being the most effective one.(7) The protein and m RNA levels of Shh, Smo and BMP-2 in the fluorosis groups(0.94±0.05, 0.89±0.07, 1.06±0.15; 0.67±0.04, 0.46±0.06, 0.77±0.06) and the control si RNA group(1.00±0.09, 0.86±0.05, 1.14±0.13; 0.61±0.04, 0.51±0.07, 0.79±0.06) were significantly increased than the blank control group(0.70±0.06, 0.69±0.06, 0.83±0.12; 0.53±0.06, 0.34±0.03, 0.69±0.06), and in the Smo si RNA group(0.52±0.03, 0.47±0.06, 0.59±0.06; 0.42±0.05, 0.28±0.06, 0.50±0.03) were significantly decreased than other groups(P<0.05).(8) Apoptosis rate in the fluorosis groups(18.49±0.80) and the control si RNA group(17.97±1.07) were significantly increased than the blank control group(11.04±1.11), and was higher in the Smo si RNA group(40.32±1.79)(P<0.05). Expression of Bax was increased in the fluorosis group(1.07±0.10) and the control si RNA group(1.19±0.12) than the blank control group(0.88±0.06). In the Smo si RNA group, expression of Bax(1.39±0.10) was the highest while expression of Bcl-2 was decreased in the fluorosis group(0.74±0.03) and the control si RNA group(0.76±0.05) than the blank control group(1.05±0.07), whereas in the Smo si RNA group expression was the lowest(0.05±0.03)(P<0.05). Conclusion:(1) Cartilage can appear different degrees of ossification and pathological changes of skeletal fluorosis when fluorine is excessive.(2) A low concentration of fluoride could promote the original generation cartilage cell proliferation in rats and a high concentration of fluoride could promote apoptosis.(3) The Hh signaling pathway, apoptosis and others factors may become pathogenesis of chondrocyte damage in chronic fluorosis. |
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