灯盏花对成骨细胞及破骨前体细胞OPG/RANKL/RANK表达的影响

Effect of Erigeron Breviscapus on the Expression of OPG/RANKL/RANK in Osteoblasts and Pre-osteoclastsin Vitro

目的研究不同浓度和不同作用时间的灯盏花对体外培养的成骨细胞和破骨前体细胞中OPG/RANKL/RANK mRNA及蛋白表达的影响,初步探讨灯盏花对骨改建的作用及其机制。方法体外培养人MG63细胞和小鼠RAW264.7细胞,分别取第三代细胞分为对照组和不同的实验组,分别应用不同浓度的灯盏花(0、0.001、0.01、0.1、1 mg/mL)干预48 h后提取总RNA和蛋白质,同时单独对0、1 mg组设12、24、48 h时间点提取蛋白质,采用半定量RT-PCR方法检测MG63细胞OPG mRNA和RANKL mRNA的表达以及RAW264.7细胞RANK mRNA的表达,采用Western blot法检测MG63细胞OPG蛋白和RANKL蛋白的表达以及RAW264.7细胞RANK蛋白的表达。结果灯盏花随浓度(0、0.001、0.01、0.1、1 mg/mL)增高,干预48 h后MG63细胞OPG mRNA和蛋白表达逐步降低(P<0.05);MG63细胞RANKL mRNA和蛋白表达逐步增加(P<0.05);RAW264.7细胞RANK mRNA表达增加(P<0.05),但0.1 mg/mL组较0.01 mg/mL组mRNA表达稍降低,RANK蛋白表达逐步增加(P<0.05)。1 mg/mL灯盏花干预12、24、48 h后MG63细胞OPG蛋白表达随时间逐步降低(P<0.05)、RANKL蛋白表达随时间逐步增加(P<0.05);RAW264.7细胞RANK蛋白表达随时间逐步增加(P<0.05)。结论灯盏花大致上呈剂量和时间依赖性抑制成骨细胞OPG表达,促进成骨细胞RANKL的表达和破骨前体细胞RANK的表达,灯盏花可能有促进骨吸收的作用。

Objective To study the effect of Erigeron Breviscapus （EB） at different concentra- tions and different intervention time points on the mRNA and protein expression of OPG/RANKL/RANK in MG63 osteoblast-like cells and RAW264.7 pre-osteoclast cells cultured in vitro , thus exploring roles EB played in bone rebuilding and its mechanisms. Methods MG63 osteoblast-like cells and RAW264.7 pre- osteoclast cells were cultured in vitro . The 3rd passage cells were divided into the control group and dif- ferent experimental groups. Total RNA and protein were respectively isolated from cells treated with dif- ferent concentrations of EB （0, 0.001,0.01,0.1, and 1.0 mg/mL） for 48 h. Meanwhile, the protein was extracted from 0 and 1 mg/mL EB groups at 12,24, and 48 h respectively. Expression of OPG mRNA and RANKL mRNA in MG63 osteoblast-like cells, and expression of RANK mRNA in RAW264.7 pre-osteo- clast cells were detected by semi-quantitative RT-PCR. Expression of OPG protein and RANKL protein in MG63 osteoblast-like cells, and expression of RANK protein in RAW264.7 pre-osteoclast cells were detected by Western blot. Results Along with increased EB concentration, expression of OPG mRNA and protein in MG63 osteoblast-like cells was gradually lowered （P 〈0.05） after 48-h intervention of EB, the expression of RANKL mRNA and protein in MG63 osteoblast-like gradually increased （P 〈0.05） ; the ex- pression of RANK mRNA in RAW264.7 pre-osteoclast cells increased （P 〈0.05）. But the expression of RANK mRNA was slightly lower in the 0.1 mg/mL EB group than in the 0.01 mg/mL EB group, and the expression of RANK protein in RAW264.7 pre-osteoclast cells gradually increased （P 〈 0.05）. After treatment with 1 mg/mL EB for 12, 24,48 h, the expression of OPG protein in MG63 osteoblast-like cells gradually decreased as time went by （P 〈0.05）, and the expression of RANKL protein in MG63 osteo- blast-like and RANK protein in RAW264.7 pre-osteoclast cells gradually increased （P 〈0.05）. The ex- pression of RANKL protein in RAW264.7 pre-osteoclast cells increased as time went by （P 〈0.05）. Con- clusion EB could inhibit the expression of OPG in osteoblasts in a dose-and time-dependent manner, promote the expression of RANKL in osteoblasts and the secretion of RANK in pre-osteoclast, indicating EB might play roles in promoting bone resorption.