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刘杰, 张慧霞, 韩炜, 谢荣, 郑凯.地黄苷A对成骨细胞增殖分化及MCP-1/CCR2信号通路的影响[J].湖南中医药大学学报英文版,2025,45(4):631-637.[Click to copy
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| 地黄苷A对成骨细胞增殖分化及MCP-1/CCR2信号通路的影响 |
| 刘杰,张慧霞,韩炜,谢荣,郑凯 |
| (新疆医科大学第六附属医院康复医学科, 新疆 乌鲁木齐 830002) |
| 摘要: |
| 目的 观察地黄苷A对成骨细胞增殖和分化能力的影响及其潜在分子作用机制。方法 以成骨细胞株MC3T3-E1细胞为体外细胞模型，以不同浓度（0、1、10、100 μmol/L）地黄苷A进行处理，采用CCK-8法和细胞增殖溴脱氧尿苷（BrdU）荧光标记法检测24、48、72 h后不同浓度地黄苷A对成骨细胞增殖能力的影响。将细胞分为空白组（生理盐水）、地黄苷A组（10 μmol/L）、地黄苷A+RS504393组（10 μmol/L地黄苷A+10 μg/mL RS504393）。碱性磷酸酶（ALP）检测试剂盒检测成骨细胞的ALP活性，采用RT-qPCR检测成骨细胞分化标志物骨钙素（OCN）、Runt相关转录因子2（Runx2）、成骨细胞特异性转录因子（Osx）和单核细胞趋化蛋白-1（MCP-1）、CC类趋化因子受体-2（CCR2）的mRNA表达量，Western blot法检测OCN、Runx2、Osx、MCP-1、CCR2的蛋白表达。结果 与0 μmol/L地黄苷A组、1 μmol/L地黄苷A组比较，10 μmol/L地黄苷A组在培养48、72 h的OD值及BrdU荧光表达量增高（P＜0.01），100 μmol/L地黄苷A组在培养48、72 h的OD值降低（P＜0.05）；与10 μmol/L地黄苷A组比较，100 μmol/L地黄苷A组在培养48、72 h的OD值降低（P＜0.05）。与0 μmol/L地黄苷A组、10 μmol/L地黄苷A组比较，100 μmol/L地黄苷A组BrdU荧光表达量在24、48、72 h降低（P＜0.01）。与1 μmol/L地黄苷A组比较，100 μmol/L地黄苷A组BrdU荧光表达量在48、72 h降低（P＜0.01）。与空白组比较，地黄苷A组3、7、14 d的ALP活性均增高（P＜0.01）；且随成骨细胞培养时间增长，ALP活性持续增高（P＜0.01）。与地黄苷A组比较，地黄苷A＋RS504393组3、7、14 d的ALP活性均降低（P＜0.01）；且随成骨细胞培养时间增长，ALP活性持续增高（P＜0.05）。与空白组比较，地黄苷A组OCN、Runx2、Osx、MCP-1、CCR2的mRNA和蛋白表达量均升高（P＜0.01）。与地黄苷A组比较，地黄苷A＋RS504393组OCN、Runx2、Osx、MCP-1、CCR2的mRNA和蛋白表达量均降低（P＜0.01）。结论 地黄苷A能通过激活MCP-1/CCR2信号通路进而促进成骨细胞的增殖和分化。 |
| 关键词: 地黄苷A 成骨细胞 成骨分化 单核细胞趋化蛋白-1 类趋化因子受体-2 |
| DOI：10.3969/j.issn.1674-070X.2025.04.007 |
| Received:November 03, 2024 |
| 基金项目:新疆维吾尔自治区重点研发科研项目（2022XJKJ009）。 |
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| Effects of rehmannioside A on osteoblast proliferation, differentiation, and the MCP-1/CCR2 signaling pathway |
| LIU Jie, ZHANG Huixia, HAN Wei, XIE Rong, ZHENG Kai |
| (Department of Rehabilitation Medicine, The Sixth Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830002, China) |
| Abstract: |
| Objective To observe the effects of rehmannioside A on the osteoblast proliferation, differentiation, and its potential molecular mechanisms. Methods Osteoblast cell line MC3T3-E1 was used as an in vitro model. Cells were treated with different concentrations of rehmannioside A (0, 1, 10, 100 μmol/L). The CCK-8 assay and bromodeoxyuridine (BrdU) fluorescence labeling method were employed to evaluate the effects of rehmannioside A on osteoblast proliferation at 24, 48, and 72 h post-treatment. Experimental groups were divided as follows: blank group (saline), rehmannioside A group (10 μmol/L), and rehmannioside A+RS504393 group (10 μmol/L rehmannioside A+10 μg/mL RS504393). Alkaline phosphatase (ALP) activity of osteoblasts was determined using an ALP assay kit. RT-qPCR was performed to measure mRNA expression levels of osteogenic differentiation markers—osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), osterix (Osx), monocyte chemoattractant protein-1 (MCP-1), and CC chemokine receptor-2(CCR2). Western blot analysis was used to measure protein expressions of OCN, Runx2, Osx, MCP-1, and CCR2. Results Compared with the 0 μmol/L and 1 μmol/L rehmannioside A groups, the 10 μmol/L rehmannioside A group showed increased OD values and BrdU fluorescence expressions at 48 and 72 h (P＜0.01), while the 100 μmol/L rehmannioside A group exhibited decreased OD values at 48 and 72 h (P＜0.05). Compared with the 10 μmol/L rehmannioside A group, the 100 μmol/L group had reduced OD values at 48 and 72 h (P＜0.05). Compared with the 0 μmol/L and 10 μmol/L rehmannioside A groups, the 100 μmol/L group showed decreased BrdU fluorescence expressions at 24, 48, and 72 h (P＜0.01). Compared with the 1 μmol/L group, the 100 μmol/L group exhibited reduced BrdU fluorescence expressions at 48 and 72 h (P＜0.01). Compared with the blank group, the rehmannioside A group demonstrated increased ALP activity at 3, 7, and 14 days (P＜0.01), with ALP activity progressively increasing over the prolonged culture time of osteoblasts (P＜0.01). However, compared with the rehmannioside A group, the rehmannioside A+RS504393 group showed decreased ALP activity at 3, 7, and 14 days (P＜0.01), with ALP activity progressively increasing over the prolonged culture time of osteoblasts (P＜0.05). Compared with the blank group, the rehmannioside A group exhibited elevated mRNA and protein expression levels of OCN, Runx2, Osx, MCP-1, and CCR2 (P＜0.01). Compared with the rehmannioside A group, the rehmannioside A+RS504393 group showed reduced mRNA and protein expressions of OCN, Runx2, Osx, MCP-1, and CCR2 (P＜0.01). Conclusion Rehmannioside A can promote osteoblast proliferation and differentiation by activating the MCP-1/CCR2 signaling pathway. |
| Key words: rehmannioside A osteoblasts osteogenic differentiation monocyte chemoattractant protein-1 CC chemokine receptor-2 |
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