Radiofrequency Treatment Attenuates Age-Related Changes in Dermal-Epidermal Junctions of Animal Skin

Abstract

The dermal-epidermal junction (DEJ) is essential for maintaining skin structural integrity and regulating cell survival and proliferation. Thus, DEJ rejuvenation is key for skin revitalization, particularly in age-related DEJ deterioration. Radiofrequency (RF) treatment, known for its ability to enhance collagen fiber production through thermal mechanisms and increase heat shock protein (HSP) expression, has emerged as a promising method for skin rejuvenation. Additionally, RF activates Piezo1, an ion channel implicated in macrophage polarization toward an M2 phenotype and enhanced TGF-β production. This study investigated the impact of RF treatment on HSP47 and HSP90 expression, known stimulators of DEJ protein expression. Furthermore, using in vitro and aged animal skin models, we assessed whether RF-induced Piezo1 activation and the subsequent M2 polarization could counter age-related DEJ changes. The RF treatment of H₂O₂-induced senescent keratinocytes upregulated the expression of HSP47, HSP90, TGF-β, and DEJ proteins, including collagen XVII. Similarly, the RF treatment of senescent macrophages increased Piezo1 and CD206 (M2 marker) expression. Conditioned media from RF-treated senescent macrophages enhanced the expression of TGF-β and DEJ proteins, such as nidogen and collagen IV, in senescent fibroblasts. In aged animal skin, RF treatment increased the expression of HSP47, HSP90, Piezo1, markers associated with M2 polarization, IL-10, and TGF-β. Additionally, RF treatment enhanced DEJ protein expression. Moreover, RF reduced lamina densa replication, disrupted lesions, promoted hemidesmosome formation, and increased epidermal thickness. Overall, RF treatment effectively enhanced DEJ protein expression and mitigated age-related DEJ structural changes by increasing HSP levels and activating Piezo1.

Keywords: Piezo1; aged mice skin; bipolar; dermal–epidermal junction; heat shock protein; monopolar; radiofrequency.

Figure 1

Expression of HSP47, HSP90, TGF-β, and DEJ proteins following RF treatment in senescent keratinocytes. (A) Schematic representation of non-senescent keratinocytes, senescent keratinocytes, and RF treatments administered to senescent keratinocytes. (B–H) Proteins were extracted from senescent keratinocytes following RF treatment and subjected to the following assays. (B–D) Western blot analysis of HSP47 and HSP90. (E) ELISA results for TGF-β. (F,G) Western blot analysis of collagen XVII. (H) ELISA results for collagen IV. Data are presented as mean ± SD of three independent experiments. **, p < 0.01 vs. first bar; $$, p < 0.01 vs. second bar (Mann–Whitney U test). CON, control; DEJ, dermal–epidermal junction; ELISA, enzyme-linked immunosorbent assay; GM, growth medium; HSP47, heat shock protein 47; HSP90, heat shock protein 90; MW, molecular weight; PBS, phosphate-buffered saline; RF, radiofrequency; SD, standard deviation; SnCs, senescent; TGF-β, transforming growth factor beta.

Figure 2

Regulation of M2 polarization and Piezo1, IL-10, TGF-β, SMAD2/3, and DEJ protein levels following RF treatment in senescent cells. (A–C) Western blot analysis of Piezo1 and CD206 in senescent macrophages following RF treatment. (D) ELISA results for IL-10 in senescent macrophages following RF treatment. (E) ELISA results for TGF-β in senescent fibroblasts influenced by RF-treated macrophages. (F,G) Western blot analysis of SMAD2/3 and pSMAD2/3 in senescent fibroblasts influenced by RF-treated macrophages. (H,I) ELISA results for nidogen and collagen IV in senescent fibroblasts influenced by RF-treated macrophages. Data are presented as mean ± SD of three independent experiments. **, p < 0.01 vs. first bar; $$, p < 0.01 vs. second bar (Mann–Whitney U test). CM_con, conditioned media from non-senescent macrophages; CM_RF, conditioned media from RF-treated senescent macrophages; CM_sen, conditioned media from senescent macrophages; CON, control; DEJ, dermal–epidermal junction; ELISA, enzyme-linked immunosorbent assay; IL-10, interleukin 10; MW, molecular weight; pSMAD2/3, phosphorylated SMAD2/3; RF, radiofrequency; SD, standard deviation; SnCs, senescent; TGF-β, transforming growth factor beta.

Figure 3

Regulation of HSP47, HSP90, Piezo1, CD206, and IL-10 in aged mice skin following RF treatment. Protein levels were assessed in the skin of 16-month-old (aged) mice following RF treatment. (A–F) Western blot analysis of HSP47, HSP90, Piezo1, and CD206. (G) ELISA results for IL-10. Data are presented as mean ± SD of three independent experiments. *, p < 0.05 and **, p < 0.01 vs. first bar; $, p < 0.05 and $$, p < 0.01 vs. fourth bar (Mann–Whitney U test). Moreover, 10:0, 10 monopolar pulses only; 5:5, 5 monopolar pulses followed by 5 bipolar pulses; 2:8, 2 monopolar pulses followed by 8 bipolar pulses; 0:10, 10 bipolar pulses only; BI, bipolar; ELISA, enzyme-linked immunosorbent assay; HSP47, heat shock protein 47; HSP90, heat shock protein 90; IL-10, interleukin 10; MONO, monopolar; MW, molecular weight; RF, radiofrequency; SD, standard deviation.

Figure 4

Regulation of TGF-β, SMAD2/3, and DEJ proteins in aged mice skin following RF treatment. Protein levels were assessed in aged mice skin following RF treatment. (A) ELISA results for TGF-β. (B,C) Western blot analysis of SMAD2/3 and pSMAD2/3. (D,E) Western blot analysis of collagen XVII. (F,G) ELISA results for nidogen and collagen IV. Data are presented as mean ± SD of three independent experiments. *, p < 0.05 and **, p < 0.01 vs. first bar; $, p < 0.05 and $$, p < 0.01 vs. fourth bar (Mann–Whitney U test). Moreover, 10:0, 10 monopolar pulses only; 5:5, 5 monopolar pulses followed by 5 bipolar pulses; 2:8, 2 monopolar pulses followed by 8 bipolar pulses; 0:10, 10 bipolar pulses only; BI, bipolar; ELISA, enzyme-linked immunosorbent assay; IL-10, interleukin 10; MONO, monopolar; MW, molecular weight; pSMAD2/3, phosphorylated SMAD2/3; RF, radiofrequency; SD, standard deviation; TGF-β, transforming growth factor beta.

Figure 5

Regulation of DEJ structural changes in aged mice skin following RF treatment. (A) Representative images of immunohistochemistry staining for nidogen, PAS staining, TEM, and HE staining in aged mice skin following RF treatment. The green mark represents BM with disruption in PAS staining. Also, in TEM image the green mark represents lamina disruption, magenta represents replication, and blue represents hemidesmosomes. Scale bars = 50 μm, 50 μm, 500 nm, and 100 μm, respectively. (B) Quantitative analysis of nidogen protein levels. (C) Quantification of BM disruptions (PAS staining). (D) Quantification of hemidesmosomes. (E) Quantification of lamina densa replications and (F) disruptions (TEM). (G) Quantitative measurement of epidermal thickness (H&E staining). Data are presented as mean ± SD of three independent experiments. *, p < 0.05 and **, p < 0.01 vs. first bar; $, p < 0.05 and $$, p < 0.01, vs. fourth bar (Mann–Whitney U test). Moreover, 10:0, 10 monopolar pulses only; 5:5, 5 monopolar pulses followed by 5 bipolar pulses; 2:8, 2 monopolar pulses followed by 8 bipolar pulses; 0:10, 10 bipolar pulses only; BI, bipolar; BM, basement membrane; DEJ, dermal–epidermal junction; HE, hematoxylin and eosin; IHC, immunohistochemistry; MONO, monopolar; PAS, periodic acid–Schiff; TEM, transmission electron microscopy.