The Second School of Clinical Medicine, Southern Medical University, Guangzhou, China.
Department of Rheumatology and Immunology, Guangdong Second Provincial General Hospital, No. 466, Xingangzhong Road, Guangzhou, 510317, China.
Mol Biol Rep. 2024 Mar 14;51(1):421. doi: 10.1007/s11033-024-09336-5.
Osteophyte development is a common characteristic of inflammatory skeletal diseases. Elevated osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) participates in pathological osteogenesis. Integrin-linked kinase (ILK) positively regulates the osteoblastic differentiation of osteoprogenitors, but whether the ILK blockage prevents osteophytes and its potential mechanism is still unknown. Furthermore, the low-dose tumor necrosis factor-α (TNF-α) promotes osteogenic differentiation, but a lack of study reports on the relationship between this cytokine and ILK. OSU-T315 is a small ILK inhibitor, which was used to determine the effect of ILK inhibition on osteogenesis and osteophyte formation.
The osteogenesis of BMSCs was evaluated using Alizarin red S staining, alkaline phosphatase, collagen type I alpha 2 chain, and bone gamma-carboxyglutamate protein. The expression and phosphorylation of protein were assessed through western blot. Immunofluorescence was employed to display the distribution of β-catenin. microCT, hematoxylin-eosin, and safranin O/fast green staining were utilized to observe the osteophyte formation in collagen antibody-induced arthritis mice. We found that ILK blockage significantly declined calcium deposition and osteoblastic markers in a dose- and time-dependent manner. Furthermore, it lowered osteogenesis in the TNF-α-induced inflammatory microenvironment by diminishing the effect of ILK and inactivating the Akt/ GSK-3β/ β-catenin pathway. Nuclear β-catenin was descended by OSU-T315 as well. Finally, the ILK suppression restrained osteophyte formation but not inflammation in vivo.
ILK inhibition lowered osteogenesis in TNF-α-related inflammatory conditions by deactivating the Akt/ GSK-3β/ β-catenin pathway. This may be a potential strategy to alleviate osteophyte development in addition to anti-inflammatory treatment.
骨赘的形成是炎症性骨骼疾病的一个共同特征。骨髓间充质干细胞（BMSCs）的成骨分化增加参与了病理性成骨作用。整合素连接激酶（ILK）正向调节成骨前体细胞的成骨分化，但 ILK 阻断是否预防骨赘形成及其潜在机制尚不清楚。此外，低剂量肿瘤坏死因子-α（TNF-α）促进成骨分化，但缺乏关于该细胞因子与 ILK 之间关系的研究报告。OSU-T315 是一种小的 ILK 抑制剂，用于确定 ILK 抑制对成骨和骨赘形成的影响。
通过茜素红 S 染色、碱性磷酸酶、胶原 I 型α2 链和骨γ-羧基谷氨酸蛋白评估 BMSCs 的成骨作用。通过 Western blot 评估蛋白的表达和磷酸化。免疫荧光显示β-连环蛋白的分布。微 CT、苏木精-伊红和番红 O/固绿染色用于观察胶原抗体诱导关节炎小鼠的骨赘形成。我们发现，ILK 阻断以剂量和时间依赖的方式显著降低钙沉积和成骨标志物。此外，它通过降低 ILK 的作用和失活 Akt/GSK-3β/β-连环蛋白通路来降低 TNF-α诱导的炎症微环境中的成骨作用。OSU-T315 还降低了核β-连环蛋白的水平。最后，ILK 抑制在体内抑制了骨赘形成，但没有抑制炎症。
ILK 抑制通过失活 Akt/GSK-3β/β-连环蛋白通路降低 TNF-α相关炎症条件下的成骨作用。这可能是除抗炎治疗外缓解骨赘形成的一种潜在策略。
