目的: 基于p38丝裂原活化蛋白激酶（MAPK）信号通路,探讨山柰酚（KFR）对人牙周韧带间充质干细胞（HPL-MSC）迁移能力和成骨细胞分化能力的影响。方法: 体外培养HPL-MSC细胞系,根据细胞处理方式不同,分为0 μmol/L KFR组、0.01 μmol/L KFR组、0.1 μmol/L KFR组、1 μmol/L KFR组、10 μmol/L KFR组、100 μmol/L KFR组、p38 MAPK抑制剂SB203580组（SB203580组）、0.1 μmol/L KFR + SB203580组（0.1 + SB203580组）,药物处理24 h或48 h。药物处理48 h后,进行成骨细胞诱导培养7 d。采用划痕实验、Transwell检测细胞迁移和侵袭能力,Western blotting评估成骨分化能力和p38 MAPK的活化水平,细胞计数试剂盒-8（CCK8）检测细胞活力。结果: 与0 μmol/L KFR组相比,处理24 h后的1 μmol/L KFR组细胞活力较高,而100 μmol/L KFR组细胞活力较低;处理48 h后的0.01、0.1、1 μmol/L KFR组细胞活力较高,而在100 μmol/L KFR组细胞活力较低。因此,选择0、0.01、0.1、1 μmol/L KFR组做细胞功能分析。与0 μmol/L KFR组相比,处理48 h后的0.01、0.1、1 μmol/L KFR组细胞迁移率及成骨桥蛋白（OPN）、骨唾液蛋白II（BSP-II）、骨形态发生蛋白2（BMP2）、骨碱性磷酸酶（ALP）、Runt相关转录因子2（RUNX2）、osterix（OSX）蛋白相对表达水平较高,0.1、1 μmol/L KFR组侵袭细胞数较多。此外,磷酸化的p38（p-p38）蛋白相对表达水平在0.01、0.1、1 μmol/L KFR组显著高于0 μmol/L KFR组,在0.1 μmol/L KFR组高于0.1 + SB203580组,而在SB203580组低于0.1 + SB203580组。与0.1 + SB203580组相比,细胞迁移率及OPN、BSP-II、BMP2、ALP、RUNX2、OSX蛋白相对表达水平在0.1 μmol/L KFR组较高,而在SB203580组较低,侵袭细胞数在0.1 μmol/L KFR组较多,而在SB203580组较少。结论: KFR能促进HPL-MSC迁移和成骨细胞分化,可能是通过激活p38 MAPK通路实现的。
Abstract
Objective To investigate the effect of kaempferol (KFR) in the migration and osteoblast differentiation of human periodontal ligament-derived mesenchymal stem cells (HPL-MSC) on the basis of p38 mitogen-activated protein kinase (MAPK) signaling pathway. Methods HPL-MSC cell line was cultured in vitro and divided into several groups: KFR group (0, 0.01, 0.1, 1, 10, 100 μmol/L KFR treatment) , p38 MAPK inhibitor SB203580 group (SB203580 group; 10 μmol/L SB203580 treatment) , 0.1 μmol/L KFR + SB203580 group (0.1 + SB203580 group; 0.1 μmol/L KFR and 10 μmol/L SB203580 co-treatment) , and drug treatment was for 24 h or 48 h. After treatment for 48 h, cells were incubated in the osteoblast inducible medium for 7 days. Cell migration and invasion ability was determined by scratch wound and Transwell assays, and osteoblast differentiation and p38 MAPK activation levels were evaluated by Western blotting. Cell viability was assessed by cell counting kit-8 (CCK8) . Results Compared with the 0 μmol/L KFR group, after 24 h treatment, cell viability of the 1 μmol/L KFR group was higher, while that of the 100 μmol/L KFR group was lower; after 48 h treatment, cell viability of the 0.01, 0.1 and 1 μmol/L KFR groups was higher, while that of the 100 μmol/L KFR group was lower. Therefore, the 0, 0.01, 0.1 and 1 μmol/L KFR groups were selected for cell function analysis. Compared with the 0 μmol/L KFR group, after 48 h treatment, cell migration rate and the relative expression levels of osteopontin (OPN) , bone salivary protein II (BSP-II) , bone morphogenetic protein 2 (BMP2) , bone alkaline phosphatase (ALP) , Runt-related transcription factor 2 (RUNX2) and osterix (OSX) in the 0.01, 0.1 and 1 μmol/L KFR group were higher, and invasive cells in the 0.1 and 1 μmol/L KFR groups were increased. In addition, relative expression level of phosphorylated p38 (p-p38) protein in the 0.01, 0.1 and 1 μmol/L KFR group was significantly higher than the 0 μmol/L KFR group, and that in the 0.1 μmol/L KFR group was higher than the 0.1 + SB203580 group, and that in the SB203580 group was lower than the 0.1 + SB203580 group. Compared with the 0.1 + SB203580 group, cell migration rate and the relative expression levels of OPN, BSP-II, BMP2, ALP, RUNX2 and OSX were higher in the 0.1 μmol/L KFR group, but lower in the SB203580 group, and invasive cells were higher in the 0.1 μmol/L KFR group, but lower in the SB203580 group. Conclusion KFR can promote the migration and osteogenic differentiation of HPL-MSC, which may be achieved by activating the p38 MAPK pathway.
关键词
山柰酚 /
人牙周韧带间充质干细胞 /
成骨分化 /
迁移 /
p38丝裂原活化蛋白激酶
Key words
kaempferol /
human periodontal ligament-derived mesenchymal stem cells /
osteoblastic differentiation /
migration /
p38 mitogen-activated protein kinase
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基金
2021年度河北省医学科学研究课题计划（20210670）
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