Lee Gwangchul, Kim Bora, Ko Youngjong, Park Mineon, Kim Donghwi, Ryu Kang Hyeon, Jun Yong Cheol, Sohn Hong Moon, Lim Wonbong
1 Department of Orthopedic Surgery, Chosun University Hospital , Gwangju, Korea.
2 Department of Premedical Science, College of Medicine, Chosun University , Gwangju, Korea.
Photomed Laser Surg. 2017 Feb;35(2):78-86. doi: 10.1089/pho.2016.4134. Epub 2016 Sep 14.
This study was designed to investigate the effect of 635-nm irradiation from a light-emitting diode (LED) on osteoclastogenesis in receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-stimulated mouse bone marrow-derived macrophages (BMMs). We determined whether 635-nm irradiation modulated the RANKL-induced osteoclastic signaling pathway in heat shock protein-27 (HSP27)-silenced cells and analyzed the functional cross talk between these factors in osteoclastic differentiation and activation.
HSP27, a member of the small HSP family, regulates oxidative stress. Clinical reports suggest that low-level laser therapy or LED therapy (LEDT) could be an effective alternative treatment for osteolytic bone disease.
In control or HSP27-siRNA-treated BMMs, the effects of LED irradiation with 635 nm and 5 mW/cm on RANKL-induced osteoclastic differentiation and activity were assessed by measuring tartrate-resistant acid phosphatase (TRAP) and resorption pit formation. Quantitative real-time polymerase chain reaction and western blot assays were carried out to assess the mRNA expression of osteoclastogenesis-related genes and phosphorylation of c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), AKT, and p38, respectively. Intracellular reactive oxygen species (ROS) generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (HDCF-DA) detection method.
The 635-nm irradiation treatment significantly increased HSP27 expression and decreased intracellular ROS generation, as well as p38 and AKT phosphorylation, leading to reductions in the expression of c-fos, NFATc1, and DC-STAMP and TRAP activation and osteoclastic bone resorption in RANKL-induced BMMs. However, in HSP27-silenced BMMs, no change was observed.
Thus, 635-nm irradiation modulates RANKL-induced osteoclastogenesis via HSP27 in BMMs. Thus, HSP27 may play a role in regulating the osteoclastic response to LEDT.
本研究旨在探讨发光二极管（LED）发出的635nm光照对核因子κB（NF-κB）配体（RANKL）刺激的小鼠骨髓来源巨噬细胞（BMMs）破骨细胞生成的影响。我们确定635nm光照是否调节热休克蛋白27（HSP27）沉默细胞中RANKL诱导的破骨细胞信号通路，并分析这些因素在破骨细胞分化和激活中的功能相互作用。
HSP27是小热休克蛋白家族的成员，可调节氧化应激。临床报告表明，低强度激光治疗或LED治疗（LEDT）可能是溶骨性骨病的一种有效替代治疗方法。
在对照或HSP27-siRNA处理的BMMs中，通过测量抗酒石酸酸性磷酸酶（TRAP）和吸收陷窝形成，评估635nm、5mW/cm的LED照射对RANKL诱导的破骨细胞分化和活性的影响。分别进行定量实时聚合酶链反应和蛋白质印迹分析，以评估破骨细胞生成相关基因的mRNA表达以及c-Jun氨基末端激酶（JNK）、细胞外信号调节激酶1/2（ERK1/2）、AKT和p38的磷酸化。使用2',7'-二氯二氢荧光素二乙酸酯（HDCF-DA）检测方法测量细胞内活性氧（ROS）的产生。
635nm光照处理显著增加HSP27表达，减少细胞内ROS产生，以及p38和AKT磷酸化，导致RANKL诱导的BMMs中c-fos、NFATc1和DC-STAMP表达降低，TRAP活性和破骨细胞骨吸收减少。然而，在HSP27沉默的BMMs中，未观察到变化。
因此，635nm光照通过HSP27调节BMMs中RANKL诱导的破骨细胞生成。因此，HSP27可能在调节破骨细胞对LEDT的反应中发挥作用。
