Beneficial effect of resveratrol on phenotypic features and activity of osteoarthritic osteoblasts.
Beneficial effect of resveratrol on phenotypic features and activity of osteoarthritic osteoblasts.
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DOI：
10.1186/s13075-017-1365-2
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发表时间：
2017-06-30
影响因子：
4.9
通讯作者：
Lajeunesse D
中科院分区：
文献类型：
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作者：
Abed É;Delalandre A;Lajeunesse D
骨关节炎（OA）是一种累及软骨下骨、关节软骨和滑膜等多个组织的复杂疾病。软骨下骨的改变包括增加但矿化不足的类骨质基质、异常成骨细胞表型，包括碱性磷酸酶（ALP）活性升高、骨钙素（OC）和转化生长因子β-1（TGF-β1）释放增加。先前的研究已经证明了OA成骨细胞（Ob）中经典Wnt信号传导（cWnt）通路的抑制。由于白藜芦醇（RSV）已被证明在不同的细胞系统中上调Wnt信号通路，我们假设RSV可能对OA Ob有益。我们使用接受全膝关节置换术的OA患者胫骨平台的软骨下骨板或尸检时正常个体的胫骨平台制备原代人Ob。采用免疫组化方法检测Sirtuin 1（Sirt 1）在正常和OA软骨下骨组织中的表达。通过qRT-PCR评价基因表达，通过蛋白质印迹分析评价蛋白质产生。分别用底物水解法和酶免疫法检测ALP活性和骨钙素分泌。茜素红染色评价矿化水平。通过使用TOPflash TCF/lef荧光素酶报告基因测定的靶基因表达和使用蛋白质印迹分析中的β-连环蛋白水平的细胞内信号传导来评估Wnt/β-连环蛋白信号传导。细胞外信号调节激酶（Erk）1/2和Smad 1/5/8通路通过蛋白质印迹分析进行评估。与正常组织相比，Sirt 1在OA软骨下骨组织中的表达和产生减少。RSV上调Sirt 1及其活性，降低瘦素的表达。RSV可增加OA Ob中Erk 1/2的磷酸化，但对Smad 1/5/8的磷酸化无影响。RSV对细胞增殖影响不大，仅轻微影响Bax/Bcl 2比值。Runx 2/Cbfa 1和过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptor，PPAR）γ的表达不受RSV剂量增加的影响。与正常Ob相比，OA Ob中观察到的ALP活性和OC释放的内源性增加仅部分校正了高RSV水平下的ALP，但未校正OC释放。相反，RSV增加OA Ob的矿化。此外，尽管Wnt 3a刺激这些细胞中的Wnt/β-连环蛋白途径，但RSV进一步增加了对Wnt 3a的反应。这些数据表明，RSV促进Sirt 1水平，抑制OA成骨细胞内源性瘦素表达，并可促进Wnt/β-catenin和Erk 1/2信号通路，这些通路在这些细胞中发生改变。
Osteoarthritis (OA) is a complex disease, which affects multiple tissues, namely the subchondral bone, articular cartilage and synovial membrane. Alterations of the subchondral bone include an increased, yet under mineralized osteoid matrix, abnormal osteoblast cell phenotype including elevated alkaline phosphatase (ALP) activity, increased release of osteocalcin (OC) and transforming growth factor β-1 (TGF-β1). Previous studies have demonstrated an inhibition of the canonical Wnt signaling (cWnt) pathway in OA osteoblasts (Ob). As resveratrol (RSV) has been shown to upregulate the Wnt signaling pathway in different cell systems, we hypothesized that RSV could be beneficial for OA Ob. We prepared primary human Ob using the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or tibial plateaus of normal individuals at autopsy. Sirtuin 1 (Sirt1) expression in normal and OA subchondral bone tissue was evaluated by immunohistochemical analysis. Expression of genes was evaluated by qRT-PCR and protein production by western blot analysis. ALP activity and osteocalcin secretion were evaluated respectively with substrate hydrolysis and enzyme immunoassay. Mineralization levels were evaluated with alizarin red staining. Wnt/β-catenin signaling was evaluated by target gene expression using the TOPflash TCF/lef luciferase reporter assay and intracellular signaling using β-catenin levels in western blot analysis. Extracellular signal-regulated kinase (Erk)1/2 and the Smad1/5/8 pathways were evaluated by western blot analysis. Sirt1 expression and production were reduced in OA subchondral bone tissue compared to normal tissue. RSV upregulated Sirt1 and its activity, and reduced the expression of leptin. RSV increased Erk1/2 phosphorylation in OA Ob; however, it had no effect on Smad 1/5/8 phosphorylation. RSV had little effect on cell proliferation and only slightly affected the Bax/Bcl2 ratio. The expression of Runx2/Cbfa1 and peroxisome proliferator-activated receptor (PPAR)γ were not affected by increasing doses of RSV. The endogenous increased ALP activity and OC release observed in OA Ob compared to normal Ob were partly corrected only for ALP at high RSV levels but not for OC release. In contrast, RSV increased the mineralization of OA Ob. Moreover, whereas Wnt3a stimulates the Wnt/β-catenin pathway in these cells, RSV further increased the response to Wnt3a. These data indicate that RSV promotes Sirt1 levels, inhibits the endogenous expression of leptin by OA osteoblasts and can promote the Wnt/β-catenin and Erk1/2 signaling pathways, which are altered in these cells.