High-Intensity Focused Ultrasound Decreases Subcutaneous Fat Tissue Thickness by Increasing Apoptosis and Autophagy.

High-intensity focused ultrasound (HIFU) leads to decreased subcutaneous adipose tissue (SAT) thickness via heat-induced adipocyte necrosis. Heat can induce adipocyte apoptosis and autophagy, and it is known that nuclear or mitochondrial p53 is involved in apoptosis and autophagy. However, whether HIFU leads to apoptosis or autophagy is unclear. We evaluated whether HIFU decreases SAT thickness via p53-related apoptosis or autophagy in high-fat diet (HFD)-fed animals. The expression of nuclear and mitochondrial p53 was increased by HIFU. HIFU also led to decreased expression of BCL2/BCL-xL (an antiapoptotic signal), increased expression of BAX/BAK (an apoptotic signal), increased levels of cleaved caspase 3/9, and increased numbers of apoptotic cells as evaluated by TUNEL assay. Furthermore, HIFU led to increased levels of ATG5, BECN1, and LC3II/LC3I, and decreased levels of p62, a marker of increased autophagy. The thickness of SAT was decreased by HIFU. In conclusion, HIFU led to nuclear and mitochondrial p53 expression, which led to apoptosis and autophagy, and eventually decreased SAT thickness in HFD-fed animals.

High-Intensity Focused Ultrasound Induces Adipogenesis via Control of Cilia in Adipose-Derived Stem Cells in Subcutaneous Adipose Tissue.

During skin aging, the volume of subcutaneous adipose tissue (sWAT) and the adipogenesis potential of adipose-derived stem cells (ASCs) decrease. It is known that the shortening of cilia length by pro-inflammatory cytokines is related to the decreased adipogenic differentiation of ASCs via increase in Wnt5a/ß-catenin. High-intensity focused ultrasound (HIFU) is known to upregulate heat shock proteins (HSP), which decrease levels of pro-inflammatory cytokines. In this study, we evaluated whether HIFU modulates the cilia of ASCs by upregulating HSP70 and decreasing inflammatory cytokines. HIFU was applied at 0.2 J to rat skin, which was harvested at 1, 3, 7, and 28 days. All results for HIFU-applied animals were compared with control animals that were not treated. HIFU increased expression of HSP70 and decreased expression of NF-κB, IL-6, and TNF-α in sWAT. HIFU decreased the expression of cilia disassembly-related factors (AurA and HDAC9) in ASCs. Furthermore, HIFU increased the expression of cilia assembly-related factors (KIF3A and IFT88), decreased that of WNT5A/ß-catenin, and increased that of the adipogenesis markers PPARγ and CEBPα in sWAT. HIFU increased the number of adipocytes in the sWAT and the thickness of sWAT. In conclusion, HIFU could selectively increase sWAT levels by modulating the cilia of ASCs and be used for skin rejuvenation.

Combined Treatment of Monopolar and Bipolar Radiofrequency Increases Skin Elasticity by Decreasing the Accumulation of Advanced Glycated End Products in Aged Animal Skin.

It is well known that skin aging is related to the destruction of collagen and elastin fibers by metalloproteinases (MMPs). Aged fibroblasts have a decreased ability to synthesize collagen and elastin. Nuclear factor erythroid 2-related factor 2 (NRF2) involves glyoxalase (GLO) activation, which inhibits the production of advanced glycated end products (AGE) and the expression of its receptor (RAGE). RAGE increases nuclear transcription factor-kappa B (NF-κB), which upregulates MMPs and decreases skin elasticity. NRF2 also decreases M1 macrophages, which secrete tumor necrosis factor-alpha (TNF-α), thereby decreasing AGE production. It is well known that radiofrequency (RF) decreases skin elasticity by increasing collagen synthesis. We evaluated whether RF increases skin elasticity via NRF2/GLO and whether they decrease AGE and RAGE expression in aged animal skin. We also compared the effects of RF based on the modes (monopolar or bipolar) or the combination used. In aged skin, NRF2, GLO-1, and M2 macrophage expression was decreased, and their expression increased when RF was applied. M1 and TNF-α demonstrated increased expression in the aged skin and decreased expression after RF application. AGE accumulation and RAGE, NF-κB, and MMP2/3/9 expression were increased in the aged skin, and they were decreased by RF. The papillary and reticular fibroblast markers showed decreased expression in young skin and increased expression in aged skin. The densities of collagen and elastin fiber in the aged skin were low, and they were increased by RF. In conclusion, RF leads to increased collagen and elastin fibers by increasing NRF2/GLO-1 and modulating M1/M2 polarization, which leads to decreased AGE and RAGE and, consequently, decreased NF-κB, which eventually slows collagen and elastin destruction. RF also leads to increased collagen and elastin fiber synthesis by increasing papillary and reticular fibroblast expression.

Radiofrequency Irradiation Mitigated UV-B-Induced Skin Pigmentation by Increasing Lymphangiogenesis.

Dermal macrophages containing melanin increase skin pigmentation since dermal melanin removal is slower than epidermal melanin removal. Lymphatic vessels are also involved in melanin clearance. We evaluated whether radiofrequency (RF) irradiation induced an increase in HSP90, which promotes lymphangiogenesis by activating the BRAF/MEK/ERK pathway and decreasing tyrosinase activity, in the UV-B exposed animal model. The HSP90/BRAF/MEK/ERK pathway was upregulated by RF. Tyrosinase activity and the VEGF-C/VEGFR 3/PI3K/pAKT1/2/pERK1/2 pathway, which increase lymphangiogenesis, as well as the expression of the lymphatic endothelial marker LYVE-1, were increased by RF. Additionally, the number of melanin-containing dermal macrophages, the melanin content in the lymph nodes, and melanin deposition in the skin were decreased by RF. In conclusion, RF increased HSP90/BRAF/MEK/ERK expression, which decreased tyrosinase activity and increased lymphangiogenesis to eventually promote the clearance of dermal melanin-containing macrophages, thereby decreasing skin pigmentation.

Attenuation Effect of Radiofrequency Irradiation on UV-B-Induced Skin Pigmentation by Decreasing Melanin Synthesis and through Upregulation of Heat Shock Protein 70.

Excess melanin deposition in the skin causes cosmetic problems. HSP70 upregulation decreases microphthalmia-associated transcription factor (MITF) expression, which eventually decreases tyrosinase activity and melanogenesis. Ultraviolet (UV) radiation upregulates p53, which increases the melanocortin receptor (MC1R) and MITF. Furthermore, HSP70 decreases p53 and radiofrequency irradiation (RF) increases HSP70. We evaluated whether RF increased HSP70 and decreased p53, consequently decreasing the MITF/tyrosinase pathway and melanogenesis in UV-B radiated animal skin. Various RF combinations with 50, 100, and 150 ms and 5, 10, and 15 W were performed on the UV-B radiated mouse skin every 2 d for 28 d. When RF was performed with 100 ms/10 W, melanin deposition, evaluated by Fontana-Masson staining, decreased without skin crust formation in the UV-B radiated skin. Thus, we evaluated the effect of RF on decreasing melanogenesis in the HEMn and UV-B radiated skin at a setting of 100 ms/10 W. HSP70 expression was decreased in the UV-B radiated skin but was increased by RF. The expression of p53, MC1R, and MITF increased in the UV-B radiated skin but was decreased by RF. The expression of p53, MC1R, and MITF increased in the α-MSH treated HEMn but was decreased by RF. The decreasing effects of RF on p53, MC1R, CREB and MITF were higher than those of HSP70-overexpressed HEMn. The decreasing effect of RF on p53, MC1R, CREB, and MITF disappeared in the HSP70-silenced HEMn. MC1R, CREB, and MITF were not significantly decreased by the p53 inhibitor in α-MSH treated HEMn. RF induced a greater decrease in MC1R, CREB, and MITF than the p53 inhibitor. Therefore, RF may have decreased melanin synthesis by increasing HSP70 and decreasing p53, thus decreasing MC1R/CREB/MITF and tyrosinase activity.

Evaluating Whether Radiofrequency Irradiation Attenuated UV-B-Induced Skin Pigmentation by Increasing Melanosomal Autophagy and Decreasing Melanin Synthesis.

Autophagy is involved in the degradation of melanosomes and the determination of skin color. TLR4 and tumor necrosis factor (TNF) signaling upregulates NF-kB expression, which is involved in the upregulation of mTOR. The activation of mTOR by UV-B exposure results in decreased autophagy, whereas radiofrequency (RF) irradiation decreases TLR4 and TNF receptor (TNFR) expression. We evaluated whether RF decreased skin pigmentation by restoring autophagy by decreasing the expression of TLR4 or TNFR/NF-κB/mTOR in the UV-B-irradiated animal model. UV-B radiation induced the expressions of TNFR, TLR, and NF-κB in the skin, which were all decreased by RF irradiation. RF irradiation also decreased phosphorylated mTOR expression and upregulated autophagy initiation factors such as FIP200, ULK1, ULK2, ATG13, and ATG101 in the UV-B-irradiated skin. Beclin 1 expression and the expression ratio of LC3-I to LC3-II were increased by UV-B/RF irradiation. Furthermore, melanin-containing autophagosomes increased with RF irradiation. Fontana-Masson staining showed that the amount of melanin deposition in the skin was decreased by RF irradiation. This study showed that RF irradiation decreased skin pigmentation by restoring melanosomal autophagy, and that the possible signal pathways which modulate autophagy could be TLR4, TNFR, NF-κB, and mTOR.

Radiofrequency Irradiation Modulates TRPV1-Related Burning Sensation in Rosacea.

Rosacea is a skin inflammatory condition that is accompanied by not only redness and flushing but also unseen symptoms, such as burning, stinging, and itching. TRPV1 expression in UVB-exposed skin can lead to a painful burning sensation. Upregulated TRPV1 expression helps release neuropeptides, including calcitonin gene-related peptide, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal peptide, which can activate macrophage and inflammatory molecules. In this study, we found that radiofrequency (RF) irradiation reduced TRPV1 activation and neuropeptide expression in a UVB-exposed in vivo model and UVB- or heat-treated in an in vitro model. RF irradiation attenuated neuropeptide-induced macrophage activation and inflammatory molecule expression. Interestingly, the burning sensation in the skin of UVB-exposed mice and patients with rosacea was significantly decreased by RF irradiation. These results can provide experimental and molecular evidence on the effective use of RF irradiation for the burning sensation in patients with rosacea.

Radiofrequency Irradiation Attenuates High-Mobility Group Box 1 and Toll-like Receptor Activation in Ultraviolet B-Induced Skin Inflammation.

Ultraviolet B (UVB) exposure activates various inflammatory molecules of keratinocytes in the epidermis layer. Such UVB-mediated skin inflammation leaves post-inflammatory hyperpigmentation (PIH). Reports show a close relationship between PIH and high-mobility group box 1 (HMGB1) and its receptors. General clinical treatments of PIH, such as oral medication and laser treatment, have reported side effects. Recent studies reported the effects of radiofrequency (RF) irradiation on restoring dermal collagen, modulating the dermal vasculature, and thickening the basement membrane. To validate how RF regulates the inflammatory molecules from UVB-irradiated keratinocytes, we used UVB-radiated keratinocytes and macrophages, as well as animal skin. In addition, we examined two cases of RF-irradiated skin inflammatory diseases. We validated the effects of RF irradiation on keratinocytes by measuring expression levels of HMGB1, Toll-like receptors (TLRs), and other inflammatory factors. The results show that the RF modulates UVB-radiated keratinocytes to secrete fewer inflammatory factors and also modulates the expression of macrophages from HMGB1, TLRs, and inflammatory factors. RF irradiation could alleviate inflammatory skin diseases in patients. RF irradiation can regulate the macrophage indirectly through modulating the keratinocyte and inflammatory molecules of macrophages reduced in vitro and in vivo. Although the study is limited by the low number of cases, it demonstrates that RF irradiation can regulate skin inflammation in patients.

Radiofrequency irradiation attenuates angiogenesis and inflammation in UVB-induced rosacea in mouse skin.

Rosacea is a skin inflammatory condition accompanied by cutaneous signs such as oedema, flushing, erythema, telangiectasia and pustules. Generally, rosacea is triggered by ultraviolet B (UVB) exposure. When exposed to UVB, skin epidermis thickens and produces elevated levels of pro-inflammatory cytokines, especially keratinocyte-related VEGF, a potent angiogenic factor. The upregulations of VEGF expression and its secretion promote the formation of new blood vessels and exacerbates rosacea. In this study, radiofrequency (RF) irradiation reduced keratinocyte proliferation in the epidermal layer, the expressions of pro-inflammatory cytokines, angiogenesis-related inflammatory factors and VEGF in our UVB-induced model of rosacea in vitro and in vivo. RF irradiation attenuated VEGF-induced angiogenesis-associated processes such as tube formation, cell migration and endothelial cell proliferation. Notably, blood vessel densities in the skins of UVB-treated mice and rosacea patients were significantly decreased by RF irradiation. These results provide experimental and molecular evidence regarding the effectiveness of RF irradiation for the treatment of rosacea.