Department of Veterinary Medical Sciences, University of Bologna, Via Tolara di Sopra 50, Ozzano dell'Emilia (Bologna) 40064, Italy.
Interdepartmental Centre for Industrial Research in Health Sciences and Technologies, University of Bologna, Via Tolara di Sopra 41/E, Ozzano dell'Emilia (Bologna) 40064, Italy.
Oxid Med Cell Longev. 2023 Aug 24;2023:7638223. doi: 10.1155/2023/7638223. eCollection 2023.
Starting from the discovery of phototherapy in the beginning of the last century, photobiomodulation (PBM) has been defined in late 1960s and, since then, widely described in different models. Robust evidence indicates that the effect of light exposure on the oxidative state of the cells and on mitochondrial dynamics, suggesting a great therapeutic potential. The translational scale-up of PBM, however, has often given contrasting and confusing results, mainly due to light exposure protocols which fail to adequately control or define factors such as emitting device features, emitted light characteristics, exposure time, cell target, and readouts. In this study, we describe the effects of a strictly controlled light-emitting diode (LED)-based PBM protocol on human fibroblasts, one of the main cells involved in skin care, regeneration, and repair. We used six emitter probes at different wavelengths (440, 525, 645, 660, 780, and 900 nm) with the same irradiance value of 0.1 mW/cm, evenly distributed over the entire surface of the cell culture well. The PBM was analyzed by three main readouts: (i) mitochondrial potential (MitoTracker Orange staining), (ii) reactive oxygen species (ROS) production (CellROX staining); and (iii) cell death (nuclear morphology). The assay was also implemented by cell-based high-content screening technology, further increasing the reliability of the data. Different exposure protocols were also tested (one, two, or three subsequent 20 s pulsed exposures at 24 hr intervals), and the 645 nm wavelength and single exposure chosen as the most efficient protocol based on the mitochondrial potential readout, further confirmed by mitochondrial fusion quantification. This protocol was then tested for its potential to prevent HO-induced oxidative stress, including modulation of the light wave frequency. Finally, we demonstrated that the controlled PBM induced by the LED light exposure generates a preconditioning stimulation of the mitochondrial potential, which protects the cell from oxidative stress damage.
从上个世纪初光疗的发现开始，光生物调节（PBM）在 20 世纪 60 年代末被定义，并在此后在不同的模型中得到了广泛描述。强有力的证据表明，光暴露对细胞的氧化状态和线粒体动力学的影响，表明其具有巨大的治疗潜力。然而，光生物调节的转化应用往往会产生相互矛盾和令人困惑的结果，主要是由于光暴露方案未能充分控制或定义发射装置特征、发射光特性、暴露时间、细胞靶标和读出等因素。在这项研究中，我们描述了一种严格控制的基于发光二极管（LED）的光生物调节方案对人类成纤维细胞的影响，人类成纤维细胞是参与皮肤护理、再生和修复的主要细胞之一。我们使用了六个不同波长（440、525、645、660、780 和 900nm）的发射器探头，辐照度值均为 0.1mW/cm2，均匀分布在整个细胞培养孔的表面。通过三种主要的读出方法分析光生物调节：（i）线粒体电位（MitoTracker Orange 染色）；（ii）活性氧（ROS）的产生（CellROX 染色）；（iii）细胞死亡（核形态）。该检测还通过基于细胞的高通量筛选技术来实现，进一步提高了数据的可靠性。还测试了不同的暴露方案（24 小时间隔的单次、两次或三次 20 秒脉冲暴露），根据线粒体电位读出值，选择 645nm 波长和单次暴露作为最有效的方案，进一步通过线粒体融合定量来确认。然后，根据其预防 HO 诱导的氧化应激的潜力来测试该方案，包括调制光波频率。最后，我们证明了 LED 光暴露引起的受控光生物调节会产生线粒体电位的预处理刺激，从而保护细胞免受氧化应激损伤。
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