Objective: To investigate the effect of zoledronate (ZOL) on osteoclast differentiation and bone resorption under high glucose, and the regulation mechanism of p38 mitogen activated kinase (p38 MAPK) signaling pathway in this process.
Methods: RAW264.7 cells were divided into four groups: low group, high group, low+ZOL group and high+ZOL group after induced into osteoclasts. Cell proliferation activity was determined by MTT assay. The migration of RAW264.7 cells were examined Optical microscopy. Immunofluorescence microscopy was used to observe the cytoskeleton and sealing zones of osteoclasts. After adding group 5: high + ZOL + SB203580 group, trap staining was used to identify the number of positive osteoclasts in each group. The number and area of resorption lacunae were observed by SEM. The mRNA and protein expression of osteoclast related factors were detected by real-time PCR and Western blotting.
Results: The cells in the 5 groups showed similar proliferative activity. High glucose promoted the migration of RAW264.7 cells (P < 0.05), inhibited the clarity of cytoskeleton and the formation of sealing zones in the osteoclasts. Exposure to high glucose significantly lowered the expressions of p38 MAPK, p-p38 MAPK, NFATc1, CTSK and TRAP, and inhibited osteoclast differentiation and bone absorption (P < 0.05). Treatment with ZOL obviously suppressed the migration ability of RAW264.7 cells, further reduced the clarity of the cytoskeleton, inhibited the formation of sealing zones of the osteoclasts, lowered the expressions of p38 MAPK, p-p38 MAPK, NFATc1, CTSK, and TRAP (P < 0.05), and inhibited osteoclast differentiation and bone absorption. Treatment with SB203580 obviously inhibited osteoclast differentiation and bone resorption and the expressions of P38 MAPK, p-p38 MAPK, NFATc1, CTSK and TRAP (P < 0.05).
Conclusions: High glucose inhibits osteoclast differentiation and bone resorption. ZOL inhibits osteoclast differentiation and bone resorption in high-glucose conditions by regulating p38 MAPK pathway, which can be a new pathway for ZOL to regulate diabetic osteoporosis.
目的: 探究在高浓度葡萄糖的条件下，唑来膦酸盐(ZOL)对破骨细胞分化及功能的影响，以及p38丝裂原蛋白活化激酶(p38 MAPK)通路在其中的调控机制。
方法: 将RAW264.7细胞向破骨细胞方向分化诱导，分为4组：低糖组(5.5 mmol/L葡萄糖)、高糖组(16.5 mmol/L葡萄糖)、低糖+ZOL组(5.5 mmol/L葡萄糖+0.1 μmol ZOL)、高糖+ZOL组(16.5 mmol/L葡萄糖+ 0.1 μmol ZOL)。MTT法测定各组细胞增殖活性，光学显微镜检测RAW264.7细胞的迁移数目，免疫荧光组织化学染色检测各组破骨细胞细胞骨架的清晰程度。增加第5组：高糖+ ZOL + SB203580(16.5 mmol/L葡萄糖+0.1 μmol ZOL +10 μmol SB203580)后，TRAP染色鉴定各组阳性破骨细胞数量；将RAW264.7细胞与牙本质磨片共同培养，扫描电子显微镜测定各组细胞骨吸收陷窝的数量及面积；实时荧光定量PCR(Real-time PCR)及Western blotting检测破骨细胞相关因子的mRNA和蛋白的表达情况。
结果: 细胞增殖实验中各组细胞增殖情况无明显差别。高糖条件对RAW264.7细胞的迁移具有促进作用，差异具有统计学意义(P < 0.05)；抑制细胞骨架的清晰程度及破骨细胞封闭区的形成；下调p38 MAPK、p-p38 MAPK、NFATc1、CTSK、TRAP mRNA和蛋白表达情况从而对破骨细胞的分化及功能发挥抑制作用，差异具有统计学意义(P < 0.05)。加入唑来膦酸盐后，RAW264.7细胞迁移受到抑制，差异具有统计学意义(P < 0.05)；细胞骨架的清晰程度进一步下降，破骨细胞封闭区的形成被进一步破坏破；p38 MAPK、p-p38 MAPK、NFATc1、CTSK、TRAP的mRNA和蛋白质表达降低，进一步抑制破骨细胞分化及功能，差异具有统计学意义(P < 0.05)；加入SB203580 后，破骨细胞分化和骨吸收功能受到抑制，p38 MAPK、p-p38 MAPK 表达受到抑制，下游NFATc1、CTSK、TRAP表达降低，差异具有统计学意义(P < 0.05)。
结论: 高糖条件抑制破骨细胞的分化生成和骨吸收功能。ZOL通过p38 MAPK信号通路实现了对高糖条件下破骨细胞分化及功能的抑制作用，提示p38 MAPK分子通路可作为唑来膦酸盐调控糖尿病性骨质疏松的新机制。
Keywords: high glucose; osteoclast; p38 mitogen-activated kinase; zoledronate.