Effects of fluoride on the expression of Indian hedgehog, parathyroid hormone-related peptide, smoothened protein and mRNA and on proliferation and apoptosis of chondrocytes / 中华地方病学杂志

ABSTRACT

Objective To study the proliferation and apoptosis and investigate the expression of Indian hedgehog (Ihh),parathyroid hormone-related peptide (PTHrp),smoothened (Smo) protein and mRNA in the cultured rat primary chondrocytes exposed to different doses of NaF.Methods The third generation articular chondrocytes of neonate rat were cultured in vitro and treated with 0 (control),5,10,20 and 40 mg/L of fluoride.The proliferation activities of cells at different times (24,48 and 72 h) were tested by Thiazolyl Blue Tetrazolium Bromide (MTT).The apoptosis rate was determined by flow cytometry.The expressions of protein and mRNA of Ihh,Smo and PTHrp at 48 h were determined by Western blotting and semi-quantitative RT-PCR,respectively.Results After exposed to 5 mg/L of fluoride for 24,48 and 72 h,the proliferation rates were significantly increased [(1.17 ± 0.07)％,(1.20 ±0.06)％,(1.16 ± 0.08)％] compared with those of control group [(1.10 ± 0.08)％,(1.13 ± 0.08)％,(1.15 ± 0.08)％],but the proliferation activity at 48 and 72 h in 40 mg/L group [(0.72 ± 0.11)％,(0.68 ± 0.04)％] was significantly lower than those in control group (all P ＜ 0.05).Compared with the control group,apoptosis rate of cartilage cell in fluoride treatment group increased gradually [(1.47 ± 0.05)％,(19.87 ± 3.03)％,(25.30 ± 1.28)％,(45.73 ± 4.63)％,F =123.328,P ＜ 0.01].Western blot analysis and RT-PCR results showed that the Ihh,PTHrp,Smo mRNA and protein 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 mRNA:0.77 ± 0.05 vs.0.98 ± 0.05,1.09 ±0.05,1.27 ± 0.03,1.46 ± 0.06;PTHrp mRNA:0.67 ± 0.07 vs.0.97 ± 0.05,1.07 ± 0.08,1.37 ± 0.05,1.45 ± 0.05;Smo mRNA:0.45 ± 0.03 vs.0.63 ±-0.04,0.71 ± 0.05,0.81 ± 0.01,1.00 ± 0.02,all P ＜ 0.05).Conclusions Low doses of fluoride can promote the proliferation of chondrocytes cultured in vitro,and high doses of fluoride can promote the apoptosis of chondrocytes cultured in vitro.The expression of Ihh signaling pathway RNAs and proteins of the cartilage cells are increased following increased levels of fluoride.The results suggest that fluorine has activated the Ihh signaling pathway in chondrocytes and promoted the proliferation and apoptosis processes which might be involved in chondrocytes injury.