中国骨质疏松杂志

通过MAPK/ERK1/2信号通路探讨固本增骨方对MC3T3-E1成骨分化的影响

The effect of the consolidate origin and increase bone formula on MC3T3-E1 cell osteogenic differentiation through MAPK/ERK1/2 signaling pathway

DOI：10.3969/j.issn.1006-7108.2023.11.006

中文关键词: 骨质疏松固本增骨方成骨分化 MAPK/ERK1/2信号通路

英文关键词:osteoporosis consolidate origin and increase bone formula osteogenic differentiation MAPK/ERK1/2 signaling pathway

基金项目:国家自然科学基金项目（81960877）；甘肃省高等学校产业支撑项目（2022CYZC-52）

作者	单位
宋敏1* 李凯1 王凯1 范凯2 海云翔1 李金益1 刘路1	1.甘肃中医药大学中医临床学院，甘肃兰州 730000 2.甘肃省中心医院急诊科，甘肃兰州 730050

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中文摘要:

目的探讨固本增骨方含药血清对MC3T3-E1细胞增殖分化的影响及与MAPK/ERK1/2信号通路的关系。方法制备固本增骨方含药血清干预MC3T3-E1细胞，CCK-8法观察各组细胞增殖情况。ALP试剂盒、茜素红染色检测各组细胞成骨矿化能力。根据CCK-8检测、ALP活性检测与茜素红染色结果筛选最佳含药血清浓度及干预时间；将MC3T3-E1细胞分A、B、C、D 4组，分别加入空白血清、20%含药血清、空白血清+PD0325901、20%含药血清+PD0325901，采用RT-PCR检测各组细胞中ERK1/2、Runx2、BMP-2 mRNA表达水平，免疫印迹法Western blot检测各组细胞中ERK1/2、P-ERK1/2、Runx2、BMP-2蛋白表达水平。结果与空白对照组相比，10%、15%、20%、25%含药血清组均能促进MC3T3-E1细胞增殖能力，提高ALP活性，其中20%含药血清干预48 h效果最为显著（P＜0.05）；茜素红染色后镜下观察在浓度为20%时染色区域钙结节数量最为密集；与A组相比，B组ERK1/2、Runx2、BMP-2 mRNA及ERK1/2、P-ERK1/2、Runx2、BMP-2蛋白的表达水平明显增高（P＜0.05）；加入阻断剂PD0325901后，C组与D组上述指标的表达明显降低（P＜0.05）；与D组相比，C组ERK1/2、Runx2、BMP-2 mRNA及ERK1/2、P-ERK1/2、Runx2、BMP-2蛋白的表达水平更低（P＜0.05）。结论固本增骨方含药血清能促进MC3T3-E1细胞的增殖及矿化，并上调成骨相关因子Runx2、BMP-2的表达，其作用机制可能与激活MAPK/ERK1/2通路有关。

英文摘要:

Objective To investigate the effect of the consolidate origin and increase bone formula on the proliferation and differentiation of MC3T3-E1 cells and its relationship with MAPK/ERK1/2 signaling pathway. Methods The consolidate origin and increase bone formula was prepared to interfere with MC3T3-E1 cells. Cell proliferation was observed using CCK-8 method. ALP kit and Alizarin red staining were used to detect the osteoblastic mineralization ability of each group. According to the results of CCK-8 detection, ALP activity detection, and Alizarin red staining, the optimal concentration of drug-containing serum and intervention time were selected. MC3T3-E1 cells were divided into groups A, B, C, and D, with addition of blank serum, 20% medicated serum, blank serum + PD0325901, and 20% medicated serum + PD0325901, respectively. mRNA expression levels of ERK1/2, Runx2, and BMP-2 in each group were detected with RT-PCR. The protein expression levels of ERK1/2, P-ERK1/2, Runx2, and BMP-2 in each group were detected with Western blotting. Results Compared to the blank control group, 10%, 15%, 20%, and 25% medicated serum groups promoted the proliferation of MC3T3-E1 cells and improved the activity of ALP, and 20% medicated serum group had the most significant effect on 48h intervention (P<0.05). Under the microscope after Alizarin red staining, the number of calcium nodules in the stained area was the most dense when the concentration was 20%. Compared to those in group A, mRNA levels of ERK1/2, Runx2, and BMP-2 and protein expression levels of ERK1/2, p-erk1/2, Runx2, and BMP-2 in group B increased significantly (P<0.05). After the addition of the blocker PD0325901, the expressions of the above indexes in group C and D decreased significantly (P<0.05). Compared to those in group D, mRNA levels of ERK1/2, Runx2, and BMP-2 and protein expression levels of ERK1/2, p-ERK1/2, Runx2, and BMP-2 in group C were lower (P<0.05). Conclusion The drug-containing serum of the consolidate origin and increase bone formula promotes the proliferation and mineralization of MC3T3-E1 cells, and up-regulates the expression of osteogenic factors Runx2 and BMP-2, which may be related to the activation of MAPK/ERK1/2 pathway.

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