| Objective: To investigate the expression of Ihh, Smo, PTHrp protein and m RNA in cartilage and chondrocytes, and to reveal Indian hedgehog signal pathway in chronic fluorosis function and mechanism of in vivo and in vitro. Methods: 1. The animal model was established. 36 healthy adult SD(Sprague-Dawley) rats were randomly divided into 3 groups. The control rats was provided with tap water in which the fluorine concentration was <1 mg/L, while low fluoride group and high fluoride group were provided fluoridation amount in drinking water, 5 mg/L and 50 mg/L, respectively. After six months of the experimental period, the fluoride contents of urine and bone were tested. Histological changes in cartilage were evaluated with Hematoxylin & Eeosin(H&E) staining using light microscopy. The protein expression of Ihh, Smo and PTHrp in cartilage of experimental animals was determined by immunohistochemistry(IHC). 2. The cell model was established. The articular chondrocytes of neonate rat were cultured in vitro and treated with 0(control), 5, 10, 20, 40 mg/L of fluoride. The proliferation activities of cells at different time(24, 48, 72 h) were tested by methyl thiazolyl tetrazolium(MTT). The apoptosis rate was used by flow cytometry method. The expressions of protein and m RNA of Ihh, Smo, PTHrp at 48 h were tested by western blot analysis and reverse transcription PCR, respectively. Results: 1. The model of chronic fluorosis rat was successfully established. The fluorine contents in the urine and bone in low and high-dose fluoride groups(2.34±0.21, 4.33±0.46; 98.81±9.03, 131.52±9.14) were higher than control group(1.73±0.07, 1.95±0.06)(P<0.05). 2. The articular cartilage tissues were obviously thinning and disordered arrangement with the increase of fluorine contents in the low and high fluoride group and some cells nuclear became degeneration. 3. Compared with control group, Ihh, Smo, PTHrp, Bax protein expression in the low and high fluoride group gradually increased, but the Bcl-2, Cyclin D1, PCNA protein expression in the low and high fluoride group gradually decreased, the difference was statistically significant(P<0.05). 4. The traits and phenotypic consistents of rat chondrocytes has successful obtained. Compared with control group [1.10±0.08, 1.13±0.08, 1.15±0.08], 5 mg/L group of proliferation rate in 24, 48, 72 h was obviously increases [1.17±0.07, 1.20±0.06, 1.16±0.08], but the proliferation activity at 48, 72 h in 40 mg/L group [0.72 ± 0.11, 0.68±0.04 ] were significantly lower than that in control group(P<0.05). Compared with the control group, fluoride treatment group cartilage cell apoptosis rate increased gradually correlation [(19.87±3.03)%,(25.30±1.28)%,(45.73±4.63)%, P<0.01]. 5. Western blot analysis and RT-PCR results showed that the Ihh, PTHrp, Smo protein and m RNA expression increased in the fluoride groups at 48 h [(Ihh protein: 0.77 ± 0.08 vs 0.98 ± 0.07, 1.23 ± 0.06, 1.37 ± 0.07, 1.34 ± 0.07; PTHrp protein: 0.68 ± 0.04 vs 0.89 ± 0.05, 0.83 ± 0.05, 1.29 ± 0.05, 1.16 ± 0.08; Smo protein: 0.37 ± 0.01 vs 0.64 ± 0.06, 0.67 ± 0.03, 0.96 ± 0.06, 0.69 ± 0.06. Ihh m RNA: 0.77 ± 0.05 vs 0.98 ± 0.05, 1.09 ± 0.05, 1.27 ± 0.03, 1.46 ± 0.06; PTHrp m RNA: 0.67 ± 0.07 vs 0.97 ± 0.05, 1.07 ± 0.08, 1.37 ± 0.05, 1.45 ± 0.05; Smo m RNA: 0.45 ± 0.03 vs 0.63 ± 0.04, 0.71 ± 0.05, 0.81 ± 0.01, 1.00 ± 0.02,(P<0.05)]. Conclusion 1. Excessive fluoride can cause the damage and the pathological morphological changes of articular cartilage. At the same time, low fluoride can promote the proliferation of chondrocytes cultured, and high fluoride can promote the apoptosis of chondrocytes cultured. 2. Excessive fluoride can lead to the increased expression of Ihh, Smo and PTHrp in cartilage and chondrocytes, The results suggest that the expression Ihh signal pathway, and elevated by increasing fluorine in vivo and in vitro. The Ihh signal pathway may be associated with proliferation and apoptosis, and jointly participated in the process of cartilage damage the pathogenesis of cartilage injury with chronic fluorosis. |
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