High-power femtosecond-terahertz pulse induces a wound response in mouse skin

Abstract

Terahertz (THz) technology has emerged for biomedical applications such as scanning, molecular spectroscopy, and medical imaging. Although a thorough assessment to predict potential concerns has to precede before practical utilization of THz source, the biological effect of THz radiation is not yet fully understood with scant related investigations. Here, we applied a femtosecond-terahertz (fs-THz) pulse to mouse skin to evaluate non-thermal effects of THz radiation. Analysis of the genome-wide expression profile in fs-THz-irradiated skin indicated that wound responses were predominantly mediated by transforming growth factor-beta (TGF-β) signaling pathways. We validated NFκB1- and Smad3/4-mediated transcriptional activation in fs-THz-irradiated skin by chromatin immunoprecipitation assay. Repeated fs-THz radiation delayed the closure of mouse skin punch wounds due to up-regulation of TGF-β. These findings suggest that fs-THz radiation initiate a wound-like signal in skin with increased expression of TGF-β and activation of its downstream target genes, which perturbs the wound healing process in vivo.

Figure 1. Functional characteristics of the in vivo response to fs-THz radiation.

(A) A schematic of the procedures for in vivo exposure and analysis of the effects of fs-THz radiation. (B) Differentially expressed genes (DEGs) in THz radiation-exposed skin. Green and red squares with blue lines denote acceptable filtering criteria (FCd researcP<0.05). See Table S1 for the list of DEGs. (C) Top 10 enriched biological functions associated with the 149 DEGs. Each enrichment score is represented by a horizontal orange bar. The vertical blue line indicates acceptable significance (P = 0.05). (D) Meta-analysis of the expression of 149 DEGs, compared against gene expression in mouse skin exposed to a variety of stimuli, including UV exposure (GSE15618), burn (GSE460), neutron irradiation (GSE25343), and wound (GSE23006). See Fig. S3 and Methods for more detailed information.

Figure 2. Molecular characteristics of responses to fs-THz radiation in mouse skin.

(A and B) Relative expression of 7 genes (Bmp2, Cd44, Krt6a, Lep, Serpine1, Sprr1b, and Thbs1) by real-time RT-PCR, selected from the top enriched bio-function, “healing” (wound response genes) (See Fig. 1C and Table S2), in C57BL/6J (A) and BALB/c nude mice (B). Skin of mice was exposed either to sham or THz radiation. (n = 8 in each group). (C) Expression of wound response genes in an in vivo wound model, 24 hours (h) after THz radiation (each, n = 4). (D) Immunohistochemical staining for Bmp2, Cd44, and Thbs1. The original magnification used for all images was ×100. A magnified region of staining is shown as an inset in the lower right. (A–C, Mean ± standard deviation (SD). *, P<0.05; **, P<0.01; ***, P<0.001).

Figure 3. Induction of TGF-β and transcriptional control of wound response.

(A) Enrichment analysis of DEGs in signaling pathways. The vertical blue line indicates acceptable significance (P = 0.05). “TGF-β Signaling” was analyzed as the most dominant pathway followed by pathways relating to translation initiation. (B) Expression of Tgfb1, by real-time RT-PCR, in skin from THz-irradiated C57BL/6J, BALB/c nude mice, and in an in vivo wound model. (C) Expression of wound response genes by real-time RT-PCR, in TGF-β-treated NIH 3T3 cells (each, n ≥ 3). (D) in silico mapping of the transcriptional regulation for DEGs. Analyzed TFs targeting mRNAs out of database (alphabetized clockwise in outer circle) are placed clockwise from top (12:00) in inner circle (See Table S3 for more detailed information). Smad3 and NFκB1 are highlighted. (E) Identification for TFBSs of the targeted genes regulated by Smad3 and NFκB1. Expression of target genes measured by ChIP-qPCR, in skin from THz-irradiated C57BL/6J and BALB/c nude mice, from an in vivo wound model, and in TGF-β-treated NIH3T3 cells. (B, C and E, Mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001).

Figure 4. Delayed wound healing associated with fs-THz radiation.

(A) Gross and microscopic time course photographs of skin wounds following treatment with sham or THz radiation. Upper panels: photographs are size-compensated and shown as binary images. Bottom panels: dotted lines indicate the margins between re-epithelialized skin and the original wound site. An arrow denotes strongly stained parts of scab spread in wounds. Histologic images were originally magnified at ×40 (scale bar: 500 μm). (B) Quantitative analysis of the open wound area in sham- or THz-irradiated skin (n = 10 in each group). (C) ELISA analysis for Tgfb1 in wounded skin following treatment with sham or THz radiation (each, n = 3). (D) Proposed model of wound response induced by fs-THz radiation. TGF-β signaling is activated in THz-exposed skin, which triggers transcription of wound healing-responsive genes via activation of the TFs, Smad3/4 and NFκB. (B and C, Mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001).