低氧环境中PADI4促进破骨细胞分化和骨吸收功能的研究

PADI4 promotes osteoclastogenesis and bone resorption under hypoxia conditions

为探讨低氧环境中肽酰基精氨酸脱亚胺酶4(peptidyl arginine deiminase 4,PADI4)对破骨细胞分化和功能的影响,采用巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)和核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)诱导单核巨噬细胞白血病细胞RAW264.7向破骨细胞分化,采用PADI4抑制剂Cl-amidine和PADI4-siRNA-MSN抑制PADI4表达,采用抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色试验、TRAP活性试验和骨吸收试验鉴定破骨细胞的分化和功能,用qRT-PCR和Western blotting分别检测PADI4、TRAP基因及其蛋白的表达水平。结果显示,低氧-M-CSF+RANKL组TRAP和PADI4 mRNA相对表达水平较常氧-M-CSF+RANKL组显著升高(P<0.05)。低氧-M-CSF+RANKL组的TRAP阳性细胞数[(7.33±1.37)个/HP]显著高于常氧-M-CSF+RANKL组[(3.33±1.63)个/HP,P<0.01],低氧-M-CSF+RANKL组较常氧-M-CSF+RANKL组TRAP活性显著增加(P<0.001)。低氧-M-CSF+RANKL组骨吸收陷窝数[(107.00±12.42)个/片]较常氧-M-CSF+RANKL组[(77.40±8.79)个/片]显著增加(P<0.05)。在低氧环境下,与对照组比较,M-CSF+RANKL+Cl-amidine组和M-CSF+RANKL+siRNA组TRAP mRNA表达水平、TRAP蛋白表达水平、TRAP阳性细胞数、TRAP活性、骨吸收陷窝数均显著降低(P<0.05)。该研究提示,在低氧环境中PADI4可能是促进巨噬细胞向破骨细胞分化以及骨吸收的关键分子。

To investigate the effect of peptidyl arginine deiminase 4(PADI4)under hypoxia conditions on osteoclast differentiation and function,RAW264.7 cells were treated with macrophage colony-stimulating factor(M-CSF)and receptor activator of nuclear factor-κB ligand(RANKL)to induce osteoclasts.The expression of PADI4 was inhibited by PADI4 inhibitor Cl-amidine and PADI4-siRNA-MSN.Osteoclastogenesis and function were respectively detected by tartrate-resistant acid phosphatase(TRAP)staining assay,TRAP activity measurement and bone resorption assay.PADI4 and TRAP mRNA and protein levels were detected by qRT-PCR and Western blotting respectively.The results showed that compared with the normoxia-M-CSF+RANKL group,the levels of PADI4 and TRAP mRNA in the hypoxia-M-CSF+RANKL group was significantly higher(P<0.05).The number of TRAP positive cells in the hypoxia-M-CSF+RANKL group([7.33±1.37]/HP)was higher than that in the normoxia-M-CSF+RANKL group([3.33±1.63]/HP,P<0.01),TRAP activity was significantly increased in the hypoxia-M-CSF+RANKL group(P<0.001).The number of resorption lacuna in the hypoxia-M-CSF+RANKL group([107.00±12.42]/piece)was higher than that in the normoxia-M-CSF+RANKL group([77.40±8.79]/piece,P<0.05).When the expression of PADI4 was inhibited under hypoxia conditions,the level of TRAP mRNA,the protein level of TRAP,the number of TRAP positive cells,TRAP activity and the number of resorption lacunae in the M-CSF+RANKL+Cl-amidine group and the M-CSF+RANKL+siRNA group were significantly lower than those in the control group(P<0.05).So,elevated expression of PADI4 under hypoxia conditions may promote osteoclasts differentiation and bone resorption.