Cheleschi Sara, De Palma Anna, Pecorelli Alessandra, Pascarelli Nicola Antonio, Valacchi Giuseppe, Belmonte Giuseppe, Carta Serafino, Galeazzi Mauro, Fioravanti Antonella
Department of Medicine, Surgery and Neuroscience, Rheumatology Unit, University of Siena, Policlinico Le Scotte, Viale Bracci 1, 53100 Siena, Italy.
Department of Medical Biotechnologies, University of Siena, Policlinico Le Scotte, Viale Bracci 1, 53100 Siena, Italy.
Int J Mol Sci. 2017 Jan 12;18(1):133. doi: 10.3390/ijms18010133.
Mechanical loading and hydrostatic pressure (HP) regulate chondrocytes' metabolism; however, how mechanical stimulation acts remain unclear. MicroRNAs (miRNAs) play an important role in cartilage homeostasis, mechanotransduction, and in the pathogenesis of osteoarthritis (OA). This study investigated the effects of a cyclic HP (1-5 MPa), in both normal and OA human chondrocytes, on the expression of , , , and , and of their target genes (, , , and ). Furthermore, we assessed the possible involvement of Wnt/β-catenin pathway in response to HP. Chondrocytes were exposed to HP for 3h and the evaluations were performed immediately after pressurization, and following 12, 24, and 48 h. Total RNA was extracted and used for real-time PCR. β-catenin was detected by Western blotting analysis and immunofluorescence. In OA chondrocytes, HP induced a significant increase ( < 0.01) of the expression levels of , , and , and a significant reduction ( < 0.01) of at all analyzed time points. , , and were significantly downregulated following HP, while no significant modification was found for . β-catenin levels were significantly increased ( < 0.001) in OA chondrocytes at basal conditions and significantly reduced ( < 0.01) by HP. Pressurization did not cause any significant modification in normal cells. In conclusion, in OA chondrocytes, HP restores the expression levels of some miRNAs, downregulates MMP-13, ADAMTS-5, and HDAC-4, and modulates the Wnt/β-catenin pathway activation.
机械负荷和静水压力（HP）调节软骨细胞的代谢；然而，机械刺激的作用方式仍不清楚。微小RNA（miRNA）在软骨稳态、机械转导以及骨关节炎（OA）的发病机制中发挥着重要作用。本研究调查了正常和OA人软骨细胞中循环HP（1 - 5兆帕）对 、 、 、 及其靶基因（ 、 、 、 ）表达的影响。此外，我们评估了Wnt/β-连环蛋白通路在对HP反应中的可能参与情况。将软骨细胞暴露于HP 3小时，在加压后立即以及12、24和48小时后进行评估。提取总RNA并用于实时PCR。通过蛋白质免疫印迹分析和免疫荧光检测β-连环蛋白。在OA软骨细胞中，在所有分析时间点，HP均显著增加（ < 0.01） 、 、 的表达水平，并显著降低（ < 0.01） 的表达水平。HP处理后 、 、 显著下调，而 未发现显著变化。在基础条件下，OA软骨细胞中β-连环蛋白水平显著增加（ < 0.001），而HP使其显著降低（ < 0.01）。加压对正常细胞未引起任何显著变化。总之，在OA软骨细胞中，HP恢复了一些miRNA的表达水平，下调了基质金属蛋白酶-13（MMP-13）、含血小板反应蛋白基序的解聚素样金属蛋白酶-5（ADAMTS-5）和组蛋白去乙酰化酶-4（HDAC-4），并调节Wnt/β-连环蛋白通路的激活。
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