Pro-Inflammatory effects of 1-MHz Pulsed Ultrasound in Human Keratinocytes

Low intensity pulsed ultrasound (LIPUS) of ~1-MHz frequency has been widely applied since the 1950s for both therapeutic and diagnostic tool [1]. The extensive clinical application of LIPUS suggests the need to better understand the related biological effects which are mostly linked to mechanical stress [2]. In this framework, we investigated whether mechanical energy transported by 1 MHz LIPUS could generate a mechanical stress able to modulate and induce the release of inflammatory cytokines such IL-6 in a human keratinocyte cell line (HaCaT). Human keratinocytes are often used to study epidermis homeostasis and its pathophysiology. In particular, these cells respond to both chemicals and physical agents, reporting a different production of cytokines, such as IL-1 and IL-6, in relation to the stimulus and the dose of the stimulus [3]. At first, we analysed the gene expression and protein secretion of the pro-inflammatory cytokine IL-6 by Real Time-qPCR and ELISA, respectively, at varying exposure parameters. Then, we focused on the activation of Nuclear Factor-κB (NF-κB), a pleiotropic regulator of many cellular pathways involved in cytokine production, cell-cycle regulation and apoptosis. Related effects on the cell cycle distribution and apoptosis have been also evaluated through flow cytometry analysis and by following the gene expression of Bax/Bcl-2. Our results pointed out that 1 hour exposure of 1 MHz LIPUS at spatial-peak temporal-average Intensity (Ispta) of 65 mW/cm2 (Figure 1a) promotes both significant IL-6 gene overexpression and protein secretion (Figure 1b), associated with the activation of Nuclear Factor-κB (Figure 1c). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability and cell proliferation, paving the way for further investigating the molecular mechanisms related to ultrasound exposure.