ERK1/2和p38MAPK介导BAPTA-AM抑制RANKL诱导小鼠骨髓巨噬细胞分化的机制研究

目的探讨BAPTA-AM对RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞能力的影响以及其信号通路机制的研究。方法体外分离培养小鼠骨髓巨噬细胞,建立RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞的实验模型。应用抗酒石酸酸性磷酸酶(TRAP)染色法检测RANKL诱导的小鼠破骨细胞分化和存活的能力及BAPTA-AM对其的抑制作用;采用免疫印迹法(Western blot)测定ERK1/2和p38MAPK蛋白的磷酸化水平。结果 RANKL诱导的小鼠骨髓巨噬细胞胞质内可见多核,并能分化成为破骨细胞。不同浓度的BAPTA-AM(1、2μmol·L-1)对破骨细胞的形成具有明显的抑制作用,且随着浓度增加能明显地降低TRAP阳性细胞数目。RANKL对小鼠骨髓巨噬细胞的ERK1/2和p38MAPK具有明显的激活作用,诱导胞质ERK1/2和p38MAPK磷酸化,而不同浓度的BAPTA-AM可明显地抑制这种磷酸化作用。结论 BAPTA-AM能明显地抑制RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞,这种抑制作用可能是通过ERK1/2和p38MAPK信号蛋白介导。

ERK 1/2 and p38 MAPK mediate the effect of BAPTA-AM inhibiting RANKL-induced bone marrow macrophages differentiation

Aim To explore whether ERK 1 /2 and p38 MAPK could mediate the effect of BAPTA-AM inhibiting RANKL-induced bone marrow macrophages differentiation.Methods BMMs were cultured with various concentrations of BAPTA-AM in the presence of MCSF(25 μg·L-1) and RANKL(25 μg·L-1) for 7 days,osteoclastogenic ability,osteoclast survival and the expression of phosphorylated ERK1 /2 and p38 MAPK were measured by TRAP staining and Western blot.Results BAPTA-AM inhibited osteoclastogenesis and osteoclast survival of BMMs by RANKL induction.The immunoblotting data revealed that BAPTA-AM could inhibit the phosphorylation of ERK1 /2 and p38 MAPK which were activated by RANKL.Conclusion ERK 1 /2 and p38 MAPK can mediate the effect of BAPTA-AM inhibiting osteoclastogenic ability of BMMs.This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.