Expressions of Ihh, Shh and Smo mRNA and protein in rats' bone exposed to different doses of fluoride and the significance / 中华地方病学杂志

ABSTRACT

Objective:To investigate the expression changes of Hedgehog related factors (Ihh, Shh and Smo) in bone of rats with chronic fluorosis, and the significance.Methods:Thirty-six healthy SD rats were divided to three groups with the method of random digits table by body weight (100 - 120 g), 12 rats in each group, half male and half female. The rats of control were fed with tap water (NaF < 1 mg/L), and the experimental rats were exposed to NaF (low dose fluoride group 5 mg/L, high dose fluoride group 50 mg/L) added to the drinking water to establish the chronic fluorosis model. After the rats were raised for six months, 24-hour urine samples were collected and the femoral metaphysis of the rats was taken. Urine fluoride and bone fluoride were detected by fluorin ion selective electrode method. Bone tissues were stained with hematoxylin-eosin and observed under light microscope. The content of bone alkalinephosphatase (BALP) in rats' serum was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of Ihh, Shh and Smo mRNA and protein in bone were detected by Real-time PCR and immunohistochemistry (IHC).Results:The contents of urine fluoride, bone fluoride and serum BALP were increased gradually in the control, low and high doses fluoride groups [urine fluoride (1.37 ± 0.44), (5.96 ± 0.56), (7.60 ± 0.61) mg/L; bone fluoride (306.04 ± 12.58), (652.91 ± 51.83), (1 094.11 ± 126.34) mg/kg; BALP (27.78 ± 4.09), (46.59 ± 5.75), (57.45 ± 3.99) U/L, P < 0.05]. It could observed that bone sclerosis by light microscope in low and high doses fluoride groups. The expressions of Ihh, Shh and Smo mRNA in high dose fluoride group (1.39 ± 0.36, 0.56 ± 0.23, 0.40 ± 0.15) were higher than those of the control and low dose fluoride groups (0.73 ± 0.19, 0.92 ± 0.34; 0.19 ± 0.04, 0.36 ± 0.16; 0.14 ± 0.04, 0.24 ± 0.13; P < 0.05). The expression of Shh mRNA in low dose fluoride group was higher than that of the control group ( P < 0.05). The expressions of Ihh and Smo protein in high dose fluoride group (138.89 ± 3.72, 149.29 ± 7.63) were higher than those of the control and the low dose fluoride groups (127.39 ± 2.69, 134.81 ± 3.53; 129.64 ± 12.62, 139.07 ± 9.30), and the low dose fluoride group were higher than those of the control group ( P < 0.05). The expression of Shh protein in high dose fluoride group (141.26 ± 7.49) was higher than that of the control group (130.96 ± 11.10, P < 0.05). Conclusion:The expression of Hedgehog signaling pathway related factors in bone of rats with chronic fluorosis is changed, which indicates that bone formation can be affected by activation of Hedgehog signaling pathway induced by fluoride.