木犀草素在炎症微环境下调控Wnt/β-catenin信号通路促进骨髓间充质干细胞成软骨分化的机制

doi:10.6040/j.issn.1671-7554.0.2025.0047

Abstract

Abstract: Objective To investigate the effect of luteolin on chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and the Wnt/β-catenin signaling pathway in an inflammatory microenvironment. Methods Network pharmacology was used to screen gene sets regulated by luteolin for chondrogenic differentiation, and a protein-protein interaction(PPI)network was constructed. Gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyses were performed to screen chondrogenic differentiation-related target genes, which were then subjected to molecular docking with luteolin. The cytotoxicity of luteolin on BMSCs was determined using the cell counting kit-8(CCK8)and cell proliferation assay. Rat BMSCs were divided into control group(caltured with 2 mL chondrogenic differentiation induction medium), model group ［using 2 mL of chondrogenic differentiation induction medium containing 10 ng/mL tumor necrosis factor-α(TNF-α)to construct an inflammatory microenvironment］, low-dose group(5 μmol luteolin intervention based on the model group), and high-dose group(10 μmol luteolin intervention based on the model group). Chondrogenic differentiation was induced in each group to construct micromass cartilage pellets. After embedding and sectioning, histologic staining(Safranin-O/Fast Green, Toluidine Blue, Alcian Blue)was performed. RT-qPCR was used to detect the expression of chondrogenic genes collagen type Ⅱ(COL2), Sry-box transcription factor 9(SOX9), and aggrecan(ACAN), as well as the expression of Wnt/β-catenin signaling pathway proteins including protein kinase A(PKA), cAMP response element-binding protein(CBP), glycogen synthase kinase 3β(GSK3β), β-catenin(β-CATENIN), cellular myelocytomatosis viral oncogene homolog(c-MYC), and Cyclin D1. Western blotting was used to determine the protein expression levels of these signaling molecules. Results A total of 54 target genes regulated by luteolin for chondrogenic differentiation were screened, 53 of which were involved in the construction of the PPI network. The target genes were associated with biological processes, including positive and negative regulation of the Wnt/β-catenin signaling pathway. Luteolin showed good docking with collagen type Ⅱ alpha 1 chain(COL2A1), SOX9 and ACAN. Luteolin at 5 μmol and 10 μmol showed no significant cytotoxicity. Histological staining revealed that the model group had sparser and more disorganized staining in cartilage pellets compared to the control group, which was significantly reversed by luteolin intervention. Compared with the control group, the model group had significantly lower mRNA expression of COL2, SOX9, ACAN, PKA, CBP, β-CATENIN, c-MYC, and Cyclin D1(P<0.05), while GSK3β expression was significantly higher(P<0.05). Protein expression levels of PKA, CBP, β-CATENIN, c-MYC, and Cyclin D1 in the Wnt/β-catenin pathway were significantly decreased(P<0.05), and GSK3β was increased(P<0.05)in the model group, all of which were significantly reversed by luteolin intervention. Conclusion Luteolin promotes chondrogenic differentiation of BMSCs in an inflammatory microenvironment, possibly by activating the Wnt/β-catenin signaling pathway.

Key words: Luteolin, Bone marrow mesenchymal stem cells, Chondrogenic differentiation, Inflammatory microenvironment, Wnt/β-catenin signaling pathway

CLC Number:

R274

ZHAO Canbin, SHAO Jiang, GUAN Donghui, QIN Ying, DING Qiang, GUO Liang, WANG Weiwei, CHEN Wei, YAN Xiaolong, ZENG Ping. Mechanism of luteolin regulating Wnt/β-catenin signal pathway in inflammatory microenvironment to promote chondrogenic differentiation of bone marrow mesenchymal stem cells[J].Journal of Shandong University (Health Sciences), 2025, 63(7): 11-22.

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Mechanism of luteolin regulating Wnt/β-catenin signal pathway in inflammatory microenvironment to promote chondrogenic differentiation of bone marrow mesenchymal stem cells

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