安石榴苷对钛颗粒诱导破骨细胞活化的作用

Effect of punicalagin on osteoclast activation induced by titanium particles

背景:有关安石榴苷对破骨细胞形成分化方面的研究很少,对磨损颗粒引起骨吸收类疾病影响的研究更少。目的:建立钛颗粒诱导的小鼠单核/巨噬细胞株RAW264.7向破骨细胞分化模型,观察不同浓度安石榴苷对破骨细胞增殖分化的影响。方法:将小鼠单核/巨噬细胞株RAW264.7分5组培养,分别加入培养基(空白组)、含0.1 g/L钛颗粒悬液的培养基、含0.1 g/L钛颗粒悬液+25μmol/L安石榴苷的培养基、含0.1 g/L钛颗粒悬液+50μmol/L安石榴苷的培养基、含0.1 g/L钛颗粒悬液+100μmol/L安石榴苷的培养基。CCK-8法检测培养1,3,5 d的细胞增殖活性;培养5 d后,抗酒石酸酸性磷酸酶染色检测成熟破骨细胞数量,Western blot法检测IκBα及核转录因子κB p65磷酸化水平,RT-PCR测量活化T细胞核因子c1、抗酒石酸酸性磷酸酶和基质金属蛋白酶9 mR NA的表达。结果与结论:与空白组比较,钛颗粒和不同浓度安石榴苷对RAW264.7细胞的增殖能力均无影响(P>0.05)。与空白组比较,钛颗粒组成熟破骨细胞数量、IκBα及核转录因子κB p65磷酸化水平、活化T细胞核因子c1mR NA、抗酒石酸酸性磷酸酶mR NA和基质金属蛋白酶9 mR NA表达显著升高(P<0.05,P<0.01),安石榴苷呈浓度依赖性降低上述指标表达。结果表明安石榴苷可抑制破骨细胞的形成与活化。

BACKGROUND： Currently, there are few researches on the effect of punicalagin on the formation and differentiation of osteoclasts, and fewer researches on the mechanism of bone resorption diseases induced by wear particles. OBJECTIVE： To establish a model of titanium particles induced mouse monocyte/macrophage cell line（RAW264.7） differentiating into osteoclasts and to observe the effect of different concentrations of punicalagins on osteoclast proliferation and differentiation. METHODS： Mouse monocyte/macrophage cell lines（RAW264.7） were divided into five groups, cultured in the culture medium of common（blank group）, 0.1 g/L titanium particle suspension, 0.1 g/L titanium particle suspension with 25 μmol/L punicalagins, 0.1 g/L titanium particle suspension with 50 μmol/L punicalagins, 0.1 g/L titanium particle suspension with 100 μmol/L punicalagins, respectively. The cell proliferative activity was detected by cell counting kit-8 assay at 1, 3 and 5 days. At 5 days after culture, number of osteoclasts was measured by tartrate-resistant acid phosphatase staining, the phosphorylation of IκBα and NF-κB p65 was detected by western blot assay, the mR NA expressions of nuclear factor of activated Tc1, tartrate-resistant acid phosphatase and matrix metalloproteinase-9 were measured by reverse transcription-PCR. RESULTS AND CONCLUSION： Compared with control group, titanium particles and different concentrations of punicalagin had no effect on the proliferation of RAW264.7 cells（P〉0.05）. The number of tartrate-resistant acid phosphatase staining-positive cells, the phosphorylation of IκBα and NF-κB p65 as well as the mR NA expressions of nuclear factor of activated Tc1, tartrate-resistant acid phosphatase and matrix metalloproteinase-9 were significantly increased compared with those of control group（P〈0.05,P〈0.01）. And punicalagins in a concentration-dependent manner decreased the expression of the above indicators. These results indicate that punicalagin can inhibit osteoclast formation and differentiation.