Anti-Fibrotic Effects of RF Electric Currents

Abstract

Hypertrophic scars and keloids are two different manifestations of excessive dermal fibrosis and are caused by an alteration in the normal wound-healing process. Treatment with radiofrequency (RF)-based therapies has proven to be useful in reducing hypertrophic scars. In this study, the effect of one of these radiofrequency therapies, Capacitive Resistive Electrical Transfer Therapy (CRET) on biomarkers of skin fibrosis was investigated. For this, in cultures of human myofibroblasts treated with CRET therapy or sham-treated, proliferation (XTT Assay), apoptosis (TUNEL Assay), and cell migration (Wound Closure Assay) were analyzed. Furthermore, in these cultures the expression and/or localization of extracellular matrix proteins such as α-SMA, Col I, Col III (immunofluorescence), metalloproteinases MMP1 and MMP9, MAP kinase ERK1/2, and the transcription factor NFκB were also investigated (immunoblot). The results have revealed that CRET decreases the expression of extracellular matrix proteins, modifies the expression of the metalloproteinase MMP9, and reduces the activation of NFκB with respect to controls, suggesting that this therapy could be useful for the treatment of fibrotic pathologies.

Keywords: MAP-Kinases; NFκB; extracellular matrix proteins; fibrosis; hypertrophic scar; keloids; metalloproteinase; myofibroblast; radiofrequency.

Figure 1

α-SMA, Col I and Col III expression. (A) α-SMA, Col I, and Col III protein expression of myofibroblasts at 48 h of CRET or sham treatment. Fluorescence intensity measurement per MHC channel. Data normalized over the corresponding sham-exposed controls (dashed line represents the control group: 100%). Means ± SEM of the fluorescence intensity/total nuclei of at least three experimental repeats per protein. *: 0.05 > p ≥ 0.01; Student’s t-test. (B) Inmunoblot for α-SMA. Representative blots at 48 h of CRET or sham treatment (100 µg protein/lane). GAPDH were used as loading control. C: Control. T: CRET treatment. (C) Immunofluorescence expression of α -SMA, Col I, Col III, and merged micrographs. Representative micrographs. Red: α-SMA, Green: Col I or Col III. Blue: cell nuclei. Scale bar: 20 µm.

Figure 2

XTT and TUNEL assays. (A) Proliferative and apoptotic myofibroblast valuation after 48 h of intermittent CRET treatment. Data are means ± SEM normalized over the corresponding sham-exposed controls (dashed line represents the control group: 100%). Five experimental replicates of XTT assay and three experimental replicates of TUNEL assay. *: 0.01 ≤ p < 0.05; **: 0.001 ≤ p < 0.01. Student’s t-test. (B) Representative micrographs of TUNEL+ cells at 48 h from CRET or sham stimulation. Green: TUNEL+ cells; Blue: Nuclei stained with DAPI. Scale bar: 20 µm.

Figure 3

Wound Assay. (A) Computational quantification of the gap closure calculated as the mean distance (µm) between the edges in three equidistant points of the gap, using Photoshop software. Data are means ± SD, normalized over the corresponding controls (dashed line represents the control group: 100%), of five experimental replicates per time interval. NS: p ≥ 0.05; Student’s t-test. (B) Representative micrographs of the wound at t = 0, 24 and 48 h from CRET or sham treatment (control). Scale bar: 50 µm.

Figure 4

MMPs expression. (A) Densitometry values for MMP9 and MMP1 expressions at 24 or 48 h of CRET or sham treatment. Data normalized over the corresponding sham-treatment controls (dashed line represents the control group 100%). Means ± SD of the protein/GAPDH ratios of at least four experimental repeats per protein and time interval. **: 0.001 ≤ p < 0.01. Student’s t-test. (B) Representative blots at 24 h or 48 h of CRET or sham treatment (100 µg protein/lane). GAPDH were used as loading control. C: Control. T: CRET treatment.

Figure 5

ERK1/2, p-ERK1/2, NFkB, and p-NFkB expression. (A) Densitometry values for ERK1/2, p-ERK1/2, NFkB, and p-NFkB expressions at 12 h or 24 h of CRET or sham treatment. Data normalized over the corresponding sham-exposed controls (dashed line represents the control group 100%). Means ± SD of the protein/GAPDH ratios of at least three experimental repeats per protein and time interval. **: 0.001 ≤ p < 0.01. *: 0.05 > p ≥ 0.01. Student’s t-test. (B) Representative blots at 12 h or 24 h of CRET or sham treatment (100 µg protein/lane). GAPDH were used as loading control. C: Control. T: CRET treatment.