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陆云, 胡玉, 陈军辉.乳香-没药含药血清通过p38 MAPK信号通路促进BMMSCs成软骨分化的机制研究[J].湖南中医药大学学报,2025,45(9):1653-1662[点击复制] |
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| 乳香-没药含药血清通过p38 MAPK信号通路促进BMMSCs成软骨分化的机制研究 |
| 陆云,胡玉,陈军辉 |
| (新疆医科大学第六附属医院脊柱三科尧骨与软组织肿瘤科, 新疆 乌鲁木齐 830000;新疆医科大学第六附属医院药学部, 新疆 乌鲁木齐 830000) |
| 摘要: |
| 目的 探究乳香-没药含药血清对骨髓间充质干细胞（BMMSCs）成软骨分化的影响及其作用机制。方法 采用全骨髓贴壁法从大鼠双侧股骨和胫骨中分离BMMSCs，观察其形态并通过流式细胞术进行鉴定；CCK-8实验筛选乳香-没药含药血清促进BMMSCs增殖的最佳含量；肿瘤坏死因子-α(TNF-α)诱导BMMSCs建立体外炎症微环境，并用CCK-8实验筛选其作用浓度；将BMMSCs分为空白组、TNF-α组、TNF-α+乳香-没药含药血清组和TNF-α+塞来昔布组，用10 ng/mL TNF-α、10%乳香-没药含药血清或10μmol/L塞来昔布处理BMMSCs后，CCK-8实验检测各组BMMSCs活力，TUNEL法检测各组BMMSCs凋亡，阿利新蓝染色检测各组BMMSCs中基质蛋白糖胺聚糖生成，Western blot检测软骨分化标志基因Sox9、Ⅱ型胶原（ColⅡ）、聚集蛋白聚糖（Aggrecan）及p38丝裂原活化蛋白激酶（p38 MAPK）信号通路相关蛋白表达。结果 分离的BMMSCs呈长梭形、多角形，排列整齐且呈漩涡状生长，CD44、CD90、CD105为阳性表达，CD34、CD45、HLA-DR为阴性表达，符合BMMSCs特征。2.5%、5%、10%的乳香-没药含药血清干预的BMMSCs活力升高（P<0.05）,20%、25%的乳香-没药含药血清干预的BMMSCs活力降低（P<0.05）；而5、10、15、20、25、30ng/mL的TNF-α处理的BMMSCs活力均降低（P<0.05）。与空白组比较，TNF-α组BMMSCs活力降低（P<0.05），凋亡率升高（P<0.05），阿利新蓝标记的蓝染细胞减少，细胞中Sox9、ColⅡ、Aggrecan蛋白相对表达量下调（P<0.05）,p-p38/p38、p-JNK/JNK蛋白相对表达比值升高（P<0.05）；与TNF-α组比较，TNF-α+乳香-没药含药血清组和TNF-α+塞来昔布组的BMMSCs活力升高（P<0.05），凋亡率降低（P<0.05），阿利新蓝标记的蓝染细胞增多，细胞中Sox9、ColⅡ、Aggrecan蛋白相对表达量上调（P<0.05）,p-p38/p38、p-JNK/JNK蛋白相对表达比值降低（P<0.05）。结论 乳香-没药含药血清可以促进炎症环境下BMMSCs增殖，并诱导其成软骨分化，该机制可能与调控p38 MAPK信号通路有关。 |
| 关键词: 乳香-没药含药血清 骨髓间充质干细胞 成软骨分化 肿瘤坏死因子-α p38丝裂原活化蛋白激酶 |
| DOI：10.3969/j.issn.1674-070X.2025.09.009 |
| 投稿时间：2025-07-01 |
| 基金项目:新疆医科大学第六附属医院(第六临床医学院)科研专项基金项目(LFYKYZXJJ2024016);新疆医科大学第六附属医院临床药学重点专科基金资助项目(202401)。 |
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| Mechanism of Ruxiang （Olibanum） and Moyao （Myrrha）-containing serum in promoting chondrogenic differentiation of BMMSCs through the p38 MAPK signaling pathway |
| LU Yun, HU Yu, CHEN Junhui |
| (University, Urumqi, Xinjiang 830000, China;Department of Pharmacy, The Sixth Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830000, China) |
| Abstract: |
| Objective To investigate the effects of Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum on chondrogenic differentiation of bone marrow mesenchymal stem cells（BMMSCs） and its mechanism of action. Methods The whole bone marrow adherence method was employed to isolate BMMSCs from the bilateral femur and tibia of rats, and the cell morphology was observed and identified by flow cytometry. The CCK-8 assay was used to determine the optimal concentration of Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum for promoting BMSC viability. Tumor necrosis factor-α（TNF-α） was used to induce BMMSCs to establish an in vitro inflammatory microenvironment, and its effective concentration was determined by CCK-8 assay.The BMMSCs were divided into blank group, TNF-α group, TNF-α+Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum group,and TNF-α+celecoxib group. Cells in each group were treated with 10 ng/mL TNF-α, 10% Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum, or 10 μmol/L celecoxib. After the treatment, BMSC viability was measured by CCK-8 assay, apoptosis by TUNEL staining, glycosaminoglycan deposition in the extracellular matrix by Alcian blue staining, and expression of chondrogenic differentiation markers, including Sox9, collagen Ⅱ（Col Ⅱ）, and aggrecan, as well as key proteins in the p38 mitogen-activated protein kinase（p38 MAPK） signaling pathway by Western blot. Results The isolated BMMSCs were spindle-or polygon-shaped, neatly arranged in a whirlpool-like pattern, positive for CD44, CD90, and CD105, and negative for CD34, CD45, and HLA-DR, consistent with the BMSC characteristics. The viability of BMMSCs treated with 2.5%, 5%, and 10% Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum increased（P＜0.05）, while that treated with 20% and 25% Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum decreased（P＜0.05）; the BMSC viability treated with 5, 10, 15, 20, 25, and 30 ng/mL TNF-α was reduced（P＜0.05）. Compared with the blank group, the TNF-α group showed reduced viability（P＜0.05）, increased apoptosis（P＜0.05）, and fewer Alcian blue-stained cells. Additionally, the relative protein expression levels of Sox9, Col Ⅱ, and aggrecan were downregulated（P＜0.05）, while the ratios of p-p38/p38 and p-JNK/JNK were elevated（P＜0.05）. Compared with the TNF-α group, both the TNF-α+Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum and TNF-α+celecoxib groups showed improved viability（P＜0.05）, reduced apoptosis（P＜0.05）, and increased Alcian blue-stained cells. The relative protein expression levels of Sox9, Col Ⅱ, and aggrecan were upregulated（P＜0.05）,while the ratios of p-p38/p38 and p-JNK/JNK decreased（P＜0.05）. Conclusion Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum can promote the proliferation of BMMSCs in inflammatory environments and induce their chondrogenic differentiation. This mechanism may be related to the regulation of the p38 MAPK signaling pathway. |
| Key words: Ruxiang（Olibanum） and Moyao（Myrrha）-containing serum bone marrow mesenchymal stem cells chondrogenic differentiation tumor necrosis factor-α p38 mitogen-activated protein kinase |
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