脂多糖对破骨细胞生成及骨吸收功能作用及其机制研究

Effect of lipopolysaccharide on osteoclasts formation and bone resorption function and its mechanism

目的探讨脂多糖(lipopolysaccharide,LPS)对破骨细胞生成及其骨吸收功能的作用及机制。方法取雄性C57BL/6小鼠股骨及胫骨骨髓,分离培养骨髓源巨噬细胞(bone marrow-derived macrophages,BMMs),并行流式细胞仪鉴定。取BMMs采用不同浓度LPS(0、100、200、500、1 000、2 000 ng/m L)培养后,以细胞计数试剂盒8(cell counting kit 8,CCK-8)检测不同浓度LPS对细胞活性影响。为探讨LPS对破骨细胞生成的影响,取BMMs分为巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)组、M-CSF+核因子κB受体活化因子配体(receptor activator of nuclear factorκB ligand,RANKL)组、M-CSF+RANKL+50 ng/m L LPS组、MCSF+RANKL+100 ng/m L LPS组,对应培养后行抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色观察,计算破骨细胞面积百分比。为探讨LPS对Connexin43蛋白及基因表达影响,将BMMs分别分为对照组(M-CSF+RANKL)、LPS组(M-CSF+RANKL+100 ng/m L LPS)以及对照组(M-CSF+RANKL)、50 ng/m L LPS组(MCSF+RANKL+50 ng/m L LPS)、100 ng/m L LPS组(M-CSF+RANKL+100 ng/m L LPS)培养后,行Western blot以及实时荧光定量PCR检测。为探讨LPS对破骨细胞骨吸收能力的影响,将BMMs分为M-CSF组、M-CSF+RANKL组、M-CSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组对应培养后,采用骨吸收实验检测骨吸收面积百分比。结果流式细胞仪鉴定培养细胞为BMMs。CCK-8法检测显示与其他浓度相比,100 ng/m L LPS明显促进BMMs活性(P<0.05)。TRAP染色示,M-CSF组未见破骨细胞生成;与M-CSF+RANKL组相比,MCSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组破骨细胞体积更大、细胞核更多,其中后者最显著,3组破骨细胞面积百分比差异均有统计学意义(P<0.05)。Western blot检测,LPS组Connexin43蛋白相对表达量较对照组明显提高(P<0.05);实时荧光定量PCR检测示,对照组、50 ng/m L LPS组以及100 ng/m L LPS组Connexin43基因相对表达量逐渐增加,比较差异有统计学意义(P<0.05)。骨吸收实验示,M-CSF组未形成破骨细胞骨吸收;M-CSF+RANKL组、M-CSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组骨吸收面积百分比逐渐增加,比较差异均有统计学意义(P<0.05)。结论 100 ng/m L LPS能够促进Connexin43的表达,从而使破骨细胞生成增多,骨吸收功能加强。

Objective To study the effect and mechanism of lipopolysaccharide（LPS） on osteoclasts formation and its bone resorption function. Methods Bone marrow-derived macrophages（BMMs） were extracted from the marrow of femur and tibia of 4-week-old male C57 BL/6 mice. Flow cytometry was used to detect BMMs. The effect of d ifferent concentrations of LPS（0, 100, 200, 500, 1 000, 2 000 ng/m L） on BMMs activity was examined by cell counting kit8（CCK-8） activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor（M-CSF） group, M-CSF＋receptor activator of nuclear factor κB ligand（RANKL）group, M-CSF＋RANKL＋50 ng/m L LPS group, M-CSF＋RANKL＋100 ng/m L LPS group. After the completion of culture,tartrate resistant acid phosphatase（TRAP） staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group（MCSF＋RANKL） and the LPS group（M-CSF＋RANKL＋100 ng/m L LPS）; and the control group（M-CSF＋RANKL）, 50 ng/m L LPS group（M-CSF＋RANKL＋50 ng/m L LPS）, and 100 ng/m L LPS group（M-CSF＋RANKL＋100 ng/m L LPS）. The expressions of Connexin43 m RNA and protein were detected by Western blot and real-time fluorescent quantitative PCR,respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF＋RANKL group, M-CSF＋RANKL＋50 ng/m L LPS group, and M-CSF＋RANKL＋100 ng/m L LPS group. Bone absorption test was used to detect the ratio of bone resorption area. Results The flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/m L LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/m L LPS（P〈0.05）. TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF＋RANKL group, the osteoclasts in MCSF＋RANKL＋50 ng/m L LPS group and M-CSF＋RANKL＋100 ng/m L LPS group were larger with more nuclei, while the osteoclasts in M-CSF＋RANKL＋100 ng/m L LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant（P〈0.05）. Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group（P〈0.05）. Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 m RNA in control group, 50 ng/m L LPS group, and100 ng/m L LPS group increased gradually, and the differences between groups were statistically significant（P〈0.05）. Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF＋RANKL group, M-CSF＋RANKL＋50 ng/m L LPS group, and M-CSF＋RANKL＋100 ng/m L LPS group, and the differences between groups were statistically significant（P〈0.05）. Conclusion LPS at concentration of 100 ng/m L can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.