ABSTRACT
Objective To observe the expression of the unfolded protein response especially the inositol-requiring enzyme-1 (IREl)-Xbp1 signaling pathway, and the change trend of osteogenic markers after inhibition of IREl expression through siRNA interference in osteoblasts exposed to fluoride. Methods Proliferation activity of MC3T3-E1 cells was detected by CCK-8 assay, and 0.0, 0.1, 1.0, 2.0, 8.0, 16.0, 20.0, 32.0, 64.0 mg/L groups were set up. Then representative doses of low, medium and high fluoride (2.0, 8.0, 20.0 mg/L) were selected to treat MC3T3-E1 cells and the expression of the unfolded protein response related genes and osteogenic markers [alkaline phosphatase (ALP), osteocalcin (OCN), Runx2, osterix, binding immunoglobulin protein (Bip), protein kinase-like endoplasmin reticulum kinase ( PERK ) , activated transcription factor 6 (ATF6), Xbp1] was detected by Real-time PCR. MC3T3-E1 cells were transfected with IRE1 siRNA and then exposed to fluoride, and the expression of IRE1 signaling pathway and osteogenic markers was detected by Western blotting and real-time PCR. Results CCK-8 results showed the bidirectional effect of fluoride on the activity of osteoblasts. Compared with the 0.0 mg/L group [1.00 ± 0.01 (d 1), 1.00 ± 0.02 (d 3), 1.00 ± 0.08 (d 7)], the osteoblast activity was significantly enhanced at 2.0 mg/L [1.11 ± 0.02 (d 1), 1.29 ± 0.02 (d 3)], 8.0 mg/L [1.16 ± 0.02 (d 1), 1.44 ± 0.03 (d 3), all P<0.05], while 20.0 mg/L inhibited cell activity [0.83 ± 0.01 (d 1), 0.81 ± 0.01 (d 3), 0.96 ± 0.04 (d 7), all P< 0.05]. Compared with the 0.0 mg/L group [6.86 ± 2.13 (ALP), 4.58 ± 1.52 (OCN), 2.65 ± 0.38 (Runx2), 12.48 ± 3.96 (osterix)], 2.0 mg/L significantly induced the expression of intracellular ALP (12.80 ± 3.62), Runx2 (6.61 ± 0.48) and osterix (21.42 ± 1.56), and the differences were statistically significant (all P< 0.05), while 20.0 mg/L inhibited the expression of ALP (0.88 ± 0.17), OCN (0.16 ± 0.05) and osterix (1.35 ± 0.51), and the differences were statistically significant (all P<0.05). Compared with the 0.0 mg/L group [1.36 ± 0.58 (IRE1), 0.96 ± 0.45 (Xbp1)], the expression of endoplasmic reticulum stress related genes IRE1 [14.84 ± 2.57 (2.0 mg/L), 4.10 ± 0.52 (8.0 mg/L), 5.30 ± 0.63 (20.0 mg/L)] and Xbp1 [2.62 ± 0.66 (2.0 mg/L), 1.97 ± 0.47 (20.0 mg/L)] were significantly increased in the corresponding fluoride groups (all P<0.05). After IRE1 gene knockout, compared with the control group [gene:3.25 ± 0.48 (OCN), 5.62 ± 1.86 (Runx2), 2.67 ± 0.35 (ALP); protein: 0.16 ± 0.03 (OCN), 0.34 ± 0.27 (ALP)], the gene expression of OCN [0.63 ± 0.46 (2.0 mg/L), 0.81 ± 0.36 (8.0 mg/L), 0.62 ± 0.31 (20.0 mg/L)], Runx2 [0.18 ± 0.03 (2.0 mg/L), 0.12 ± 0.01 (8.0 mg/L), 1.09 ± 0.33 (20.0 mg/L)] and ALP [1.01 ± 0.12 (8.0 mg/L), 0.38 ± 0.09 (20.0 mg/L)] in the corresponding fluoride groups were significantly decreased (all P < 0.05), protein expression of OCN [0.06 ± 0.02 (2.0 mg/L), 0.06 ± 0.02 (8.0 mg/L), 0.07 ± 0.03 (20.0 mg/L)], and ALP [0.02 ± 0.01 (8.0 mg/L), 0 (20.0 mg/L)] were significantly decreased (all P< 0.05). Conclusion Unfolded protein response is observed under different doses of fluoride in osteoblasts, and IRE1 gene knockout has inhibited the expression of ALP, OCN, osterix and Runx2 in osteoblasts induced by fluoride, which suggests that IRE1 signaling pathway may play a key role in the differentiation of osteoblasts exposed to fluoride.