Department of Internal Medicine, Division of Clinical Immunology, Erasmus Medical Center, Rotterdam, The Netherlands.
Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands.
J Cell Mol Med. 2018 Feb;22(2):873-882. doi: 10.1111/jcmm.13373. Epub 2017 Oct 3.
We recently showed that patients with primary Sjögren Syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favourable effects on BMD. To study the direct effects of HCQ on human MSC-derived osteoblast activity. Osteoblasts were cultured from human mesenchymal stromal cells (hMSCs). Cultures were treated with different HCQ doses (control, 1 and 5 µg/ml). Alkaline phosphatase activity and calcium measurements were performed to evaluate osteoblast differentiation and activity, respectively. Detailed microarray analysis was performed in 5 µg/ml HCQ-treated cells and controls followed by qPCR validation. Additional cultures were performed using the cholesterol synthesis inhibitor simvastatin (SIM) to evaluate a potential mechanism of action. We showed that HCQ inhibits both MSC-derived osteoblast differentiation and mineralization in vitro. Microarray analysis and additional PCR validation revealed a highly significant up-regulation of the cholesterol biosynthesis, lysosomal and extracellular matrix pathways in the 5 µg/ml HCQ-treated cells compared to controls. Besides, we demonstrated that 1 µM SIM also decreases MSC-derived osteoblast differentiation and mineralization compared to controls. It appears that the positive effect of HCQ on BMD cannot be explained by a stimulating effect on the MSC-derived osteoblast. The discrepancy between high BMD and decreased MSC-derived osteoblast function due to HCQ treatment might be caused by systemic factors that stimulate bone formation and/or local factors that reduce bone resorption, which is lacking in cell cultures.
我们最近表明，原发性干燥综合征（pSS）患者的骨密度（BMD）明显高于健康对照组。大多数患者（69%）正在使用羟氯喹（HCQ），这可能对 BMD 有有利影响。为了研究 HCQ 对人骨髓间充质干细胞衍生的成骨细胞活性的直接影响。从人间充质基质细胞（hMSC）培养成骨细胞。用不同剂量的 HCQ（对照、1 和 5μg/ml）处理培养物。分别通过碱性磷酸酶活性和钙测量来评估成骨细胞分化和活性。在 5μg/ml HCQ 处理的细胞和对照物中进行详细的微阵列分析，然后进行 qPCR 验证。使用胆固醇合成抑制剂辛伐他汀（SIM）进行额外的培养，以评估潜在的作用机制。我们表明 HCQ 在体外抑制 MSC 衍生的成骨细胞分化和矿化。微阵列分析和额外的 PCR 验证表明，与对照组相比，5μg/ml HCQ 处理的细胞中胆固醇生物合成、溶酶体和细胞外基质途径显著上调。此外，我们还证明 1μM SIM 也降低了 MSC 衍生的成骨细胞分化和矿化与对照组相比。似乎 HCQ 对 BMD 的积极影响不能用其对 MSC 衍生的成骨细胞的刺激作用来解释。由于 HCQ 治疗导致高 BMD 和 MSC 衍生的成骨细胞功能下降之间的差异可能是由于刺激骨形成的系统因素和/或减少骨吸收的局部因素造成的，而细胞培养中缺乏这些因素。
