Resveratrol promotes osteogenic differentiation of bone marrow mesenchymal stem cells in an inflammatory microenvironment

doi:10.12307/2026.057

Abstract

Abstract: BACKGROUND: The incidence of peri-implantitis is increasing in China, which greatly affects restorative results of implants. To explore whether resveratrol can improve the biological activity and mechanism of bone marrow mesenchymal stem cells in inflammatory microenvironment can provide some theoretical support for the development of therapeutic drugs related to peri-implantitis.
OBJECTIVE: To investigate the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells in an inflammatory microenvironment.
METHODS: Bone marrow mesenchymal stem cells were extracted and purified from SD rats by whole bone marrow apposition method. Bone marrow mesenchymal stem cells were divided into three groups as follows: normal control group, lipopolysaccharide group (1 μg/mL), lipopolysaccharide + resveratrol (1 μmol/L) group, and received the intervention for 24 hours. DCFH-DA reactive oxygen species fluorescent probe was used to detect reactive oxygen species levels. RT-qPCR was used to detect the mRNA expression of pro-inflammatory genes interleukin-1β, interleukin-18, and tumor necrosis factor-α. Alkaline phosphatase staining was performed 7 days after osteogenic induction. Alizarin red staining was performed 21 days after osteogenic induction. RT-qPCR was used to detect the mRNA expression of osteogenic genes Runx2 and Osx 7 days after osteogenic induction. Western blot assay was used to detect the protein expression of FoxO1 and NLRP3 7 days after osteogenic induction.
RESULTS AND CONCLUSION: (1) Resveratrol alleviated the inhibitory effect of the inflammatory microenvironment on alkaline phosphatase activity and calcified nodule formation in bone marrow mesenchymal stem cells. (2) Resveratrol inhibited mRNA expression of pro-inflammatory genes interleukin-1β, interleukin-18, and tumor necrosis factor-α and up-regulated mRNA expression of osteogenic genes Runx2 and Osx in bone marrow mesenchymal stem cells in the inflammatory microenvironment. (3) Resveratrol downregulated reactive oxygen species levels in bone marrow mesenchymal stem cells in the inflammatory microenvironment. (4) Resveratrol promoted FoxO1 protein expression and inhibited NLRP3 protein expression. (5) The results confirm that resveratrol (1 μmol/L) activates the FoxO1 signaling pathway and inhibits the expression of NLRP3 inflammatory vesicles, which reduces the release of inflammatory factors, removes excessive reactive oxygen species, and ultimately promotes the osteogenic differentiation of bone marrow mesenchymal stem cells in the inflammatory microenvironment.

Key words: bone marrow mesenchymal stem cell, resveratrol, lipopolysaccharide, peri-implantitis, osteogenic differentiation, FoxO1, reactive oxygen species, NLRP3

CLC Number:

Zou Yulian, Chen Chaopei, Huang Haixia, Lan Yuyan, Liu Min, Huang Ting. Resveratrol promotes osteogenic differentiation of bone marrow mesenchymal stem cells in an inflammatory microenvironment[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(7): 1669-1678.

Figures/Tables 4

2.3 大鼠BMSCs的多向分化能力大鼠BMSCs经成骨诱导培养21 d后，茜素红染色可见红染斑块即为钙结节(图3A)，证明BMSCs可被诱导成骨分化。成脂诱导培养21 d后，油红O染色可见圆形橙红染色即为脂滴(图3B)，证明BMSCs可被诱导成脂分化。 2.4 不同浓度白藜芦醇对大鼠BMSCs增殖的影响采用CCK-8法检测不同浓度(1，5，10，20，40 μmol/L) 白藜芦醇培养24 h后对BMSCs增殖活性的影响，结果见图4。如图所示，与对照组相比，当白藜芦醇浓度> 10 μmol/L时细胞增殖明显受到抑制，差异有显著性意义(P < 0.05)，说明白藜芦醇浓度超过10 μmol/L时，可对BMSCs产生毒性作用。 2.5 白藜芦醇减轻炎症微环境对大鼠BMSCs增殖的抑制采用CCK-8检测BMSCs在正常环境下、脂多糖刺激环境下以及在脂多糖刺激环境下添加不同浓度(1，5，10 μmol/L)白藜芦醇的增殖情况，见图5。实验结果显示，孵育24，36 h后，与正常对照组相比，脂多糖组BMSCs增殖明显受到抑制(P < 0.05)。与脂多糖组相比，1，5 μmol/L白藜芦醇组BMSCs增殖抑制得到缓解(P < 0.05)。孵育48 h后，脂多糖组增殖率仍低于正常对照组。与脂多糖组相比，白藜芦醇组BMSCs增殖率均升高(P < 0.05)，其中加入1 μmol/L白藜芦醇组增殖率最高。综上，1 μmol/L白藜芦醇在炎症微环境中对BMSCs的保护作用最为显著，因此选择1 μmol/L白藜芦醇用于后续实验。 2.6 白藜芦醇减轻炎症微环境中大鼠BMSCs的成骨分化障碍成骨诱导7 d后各组大鼠BMSCs碱性磷酸酶染色结果见图6。正常对照组碱性磷酸酶染色最深，脂多糖组碱性磷酸酶染色较正常对照组明显变浅，而脂多糖+白藜芦醇组碱性磷酸酶染色较脂多糖组有所加深。成骨诱导21 d后各组大鼠BMSCs茜素红染色结果见图7。正常对照组可见大面积红染钙结节，脂多糖组较正常对照组红染面积明显减少且颜色变浅，而脂多糖+白藜芦醇组红染面积较脂多糖组增加。钙化结节定量检测结果见图7C，正常对照组的吸光度值最高，说明钙化结节数量最多，与其他两组比较差异均有显著性意义(P < 0.05)。脂多糖组的吸光度值最低，说明钙化结节数量最少，与其他两组比较差异有显著性意义(P < 0.05)。脂多糖+白藜芦醇组的吸光度值高于脂多糖组，说明钙化结节数量上升，差异有显著性意义(P < 0.05)。这些结果表明，白藜芦醇可减轻炎症微环境中BMSCs的成骨分化障碍。 2.7 白藜芦醇下调炎症微环境中大鼠BMSCs内活性氧水平各组大鼠BMSCs内活性氧水平见图8，绿色荧光代表活性氧的产生，如图8A所示，正常对照组的绿色荧光最为微弱，荧光覆盖面积也最小，这表明在正常生理状态下，BMSCs内活性氧含量相对较低。而脂多糖组绿色荧光最亮，荧光面积最大，表明细胞内活性氧产生量显著增加。相比之下，脂多糖+白藜芦醇组绿色荧光强度明显减弱，荧光面积也相对减小，这提示白藜芦醇可能对脂多糖诱导的活性氧过量产生具有一定的抑制作用。为进一步量化这些观察结果，采用Image J软件对荧光显微镜图像进行半定量分析，分析结果如图8B所示，正常对照组的平均荧光强度最低，即在正常条件下，BMSCs内的活性氧水平维持在一个较低的水平，而脂多糖组的平均荧光强度最高，这进一步证实了脂多糖能够有效诱导BMSCs内活性氧的过量产生。更为重要的是，与脂多糖组相比，脂多糖+白藜芦醇组的平均荧光强度显著下降(P < 0.05)，这一结果明确表明白藜芦醇能够显著抑制由脂多糖刺激引起的BMSCs内活性氧的过量产生，从而发挥抗氧化作用。"

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