Abstract：
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：骨赘发育是炎症性骨骼疾病的共同特征。骨髓间充质干细胞（BMSCs）成骨分化的提高参与病理性成骨。整合素连接激酶（ILK）正调节骨祖细胞的成骨细胞分化，但ILK阻断是否能预防骨赘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，但缺乏关于这种细胞因子与ILK之间关系的研究报道。OSU-T315是一种小型ILK抑制剂，用于确定ILK抑制对成骨和骨赘形成的影响。
结果：用茜素红S染色评价骨髓间充质干细胞的成骨能力，碱性磷酸酶,胶原蛋白I型α2链，和骨γ-羧基谷氨酸蛋白。通过蛋白质印迹评估蛋白质的表达和磷酸化。免疫荧光用于显示β-连环蛋白的分布。microCT,苏木精-伊红,采用番红O/固绿染色观察胶原抗体诱导的关节炎小鼠骨赘形成。我们发现ILK阻断以剂量和时间依赖性方式显着降低了钙沉积和成骨细胞标志物。此外，它通过减少ILK的作用和使Akt/GSK-3β/β-catenin途径失活而降低了TNF-α诱导的炎症微环境中的骨生成。核β-连环蛋白也是OSU-T315的后代。最后,ILK抑制抑制骨赘形成，但不抑制体内炎症。
结论：ILK抑制通过使Akt/GSK-3β/β-catenin通路失活而降低了TNF-α相关炎症状态下的骨生成。除了抗炎治疗外，这可能是缓解骨赘发育的潜在策略。
结果：用茜素红S染色评价骨髓间充质干细胞的成骨能力，碱性磷酸酶,胶原蛋白I型α2链，和骨γ-羧基谷氨酸蛋白。通过蛋白质印迹评估蛋白质的表达和磷酸化。免疫荧光用于显示β-连环蛋白的分布。microCT,苏木精-伊红,采用番红O/固绿染色观察胶原抗体诱导的关节炎小鼠骨赘形成。我们发现ILK阻断以剂量和时间依赖性方式显着降低了钙沉积和成骨细胞标志物。此外，它通过减少ILK的作用和使Akt/GSK-3β/β-catenin途径失活而降低了TNF-α诱导的炎症微环境中的骨生成。核β-连环蛋白也是OSU-T315的后代。最后,ILK抑制抑制骨赘形成，但不抑制体内炎症。
结论：ILK抑制通过使Akt/GSK-3β/β-catenin通路失活而降低了TNF-α相关炎症状态下的骨生成。除了抗炎治疗外，这可能是缓解骨赘发育的潜在策略。
