Effect of fluoride on the protein expression of interleukin-6/glycoprotein 130 signaling pathway in bone tissue of mice

Abstract

Objective To observe the expression of interleukin-6 (IL-6), glycoprotein 130 (gp130), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) in bone tissue of fluorosis mice and explore the mechanisms of IL-6/gp130 signaling pathway in the pathogenesis of bone injury caused by fluoride. Methods Sixty-four male Balb/c mice were divided into 4 groups based on body weight by random number table method and 16 mice were in each group. The mice in control group were fed with distilled water, experimental animals in low, middle and high dose groups were fed with distilled water containing NaF 25, 50 and 100 mg/L, respectively. The mice were fed for three months to establish a drinking water fluorosis model. Mice dental fluorosis was observed and the fluoride contents in spine were detected by fluorine-ion selective electrode method. The changes of morphology and cell ultra structure in fluorosis bone tissue were observed by optical and electronic microscope. The activities of serum alkaline phosphatase (ALP) and acid phosphatase (ACP) were detected by micro enzyme labeled method. The levels of serum IL-6 were detected by enzyme-linked immunosorbent assay. The mRNA expression levels of IL-6, gp130, PI3K and Akt in long bone tissue were detected by real-time fluorescence quantitative PCR (real-time PCR). The protein expression levels and activity of gp130, PI3K and Akt were detected by Western blotting. Results The rates of dental fluorosis in low fluoride group, middle fluoride group and high fluoride group were 62.5% (10/16), 100.0% (15/15) and 100.0% (16/16), respectively, and all the fluoride groups were higher than control group (0, 0/15, all P < 0.05). Fluoride contents of bone were significantly increased with increase of fluoride doses (F= 309.716, P < 0.05). The bone trabecular thickness and density in fluorosis mice were remarkably increased and disorder, and bone sclerosis was observed under optical microscope. The percentage of trabecular bone area was significantly increased with increase of fluoride doses(F= 25.161, P < 0.05). Ultra structurally, swelling of cells, dilation of endoplasmic reticulum, chromatin margination in osteoblasts and osteoclasts of high fluoride group were observed which indicated that cells ultra structure was apparently damaged. The activities of serum ALP and ACP were significantly increased with increase of fluoride doses (F= 19.586, 13.329, all P < 0.05), and both high fluoride group [(8.05 ± 1.10), (10.95 ± 1.85) King unit] and middle fluoride group [(6.48 ± 0.92), (9.18 ± 0.76) King unit] were significantly higher than control group [(5.15 ± 0.73), (7.49 ± 0.66) King unit, all P < 0.05]. The levels of serum IL-6 in control group, low fluoride group, middle fluoride group and high fluoride group were (5.98 ± 1.43), (7.54 ± 2.16), (5.25 ± 1.97), (6.31 ± 1.36) ng/L, respectively, the differences were statistically significant (F= 3.840, P < 0.05), and low fluoride group was significantly higher than control group (P < 0.05), and middle fluoride group was significantly lower than low fluoride group (P < 0.05). The differences of mRNA expression levels of IL-6, gp130, PI3K and Akt in long bone tissue between groups were statistically significant (F= 9.952, 8.954, 6.997, 21.504, all P < 0.05), and high fluoride group (0.59 ± 0.42, 0.46 ± 0.22, 0.55 ± 0.30, 0.61 ± 0.10) was lower than control group (1.10 ± 0.54, 1.16 ± 0.67, 1.20 ± 0.84, 1.06 ± 0.37) and low fluoride group (1.86 ± 0.63, 1.61 ± 0.64, 1.74 ± 0.65, 1.70 ± 0.58, all P < 0.05), and all mRNA expression except gp130 of low fluoride group was higher than that of control group (all P < 0.05). The differences of protein expression levels and activities of gp130, PI3K and Akt in long bone tissue between groups were statistically significant (F= 4.777, 7.479, 3.535, all P < 0.05), and low fluoride group (0.66 ± 0.10, 0.69 ± 0.12, 0.92 ± 0.09) was significantly higher than control group (0.51 ± 0.11, 0.47 ± 0.06, 0.66 ± 0.14, all P < 0.05), and high fluoride group (0.42 ± 0.11, 0.38 ± 0.06, 0.67 ± 0.14) was lower than low fluoride group (all P < 0.05). Conclusion The mRNA and protein expression levels of IL-6, gp130, PI3K and Akt in bone tissue of fluorosis mice have changed, which indicates that IL-6/gp130 signaling pathway may be involved in the pathogenesis of bone injury caused by fluoride. Key words: Fluorine; Mice; Interleukin-6; Glycoproteins; Bone and Bones