The effect of Q-switched 1064-nm dymium-doped yttrium aluminum garnet laser on the skin barrier and collagen synthesis through miR-24-3p

Abstract

Due to the increase of the world's population aging, how to restore youthfulness to the skin has attracted much attention. It is well known that collagen synthesis and changes in skin barrier play an important role in the process of skin aging. However, whether Q-switched 1064-nm Nd:YAG laser (1064-QSNYL) determines the involvement of miRNAs in skin collagen synthesis and skin barrier changes remains to be elucidated. Upstream miRNAs of p38 molecular pathway have been predicted by bioinformatic database and the relationship between miRNAs and p38 verified by dual-luciferase reporter gene and Western blotting. RT-qPCR analysis detected the expression of miR-24-3p and mRNA for collagen and skin barrier-related molecules, such as keratin 10 (K10), filaggrin, and Aquaporin 4 (APQ4), in mice back skin and in the keratinocyte cell line HaCaT. Western blotting and immunofluorescence (IF) have been used to detect collagen expression and to localize, as well as quantify K10, filaggrin, and APQ4, respectively. In this study, we show that p38 is the main target gene of miRNA-24-3p, and laser irradiation at 1.5 J/cm² inhibits miR-24-3p expression. Irradiation treatment upregulates the moisture, elasticity, hydroxyproline, and superoxide dismutase content of mice skin, as well as inhibits trans-epidermal water loss. Irradiation also increases collagen, K10, filaggrin, and APQ4 in both mice skin and HaCaT cells. Interestingly, we found that miR-24-3p overexpression inhibits the effect of irradiation on collagen synthesis and skin barrier. We show for the first time that 1064-QSNYL promotes collagen synthesis and protective effects on skin barrier by downregulating miR-24-3p.

Keywords: 1064-nm Q-switched neodymium-doped yttrium aluminum garnet laser; Collagen; Skin barrier; miR-24-3p.

Fig. 1

1064-QSNYL upregulated the expression level of p38, whereas miR-24-3p targeted and negatively regulated p38. a Effects of laser irradiation on the protein expressions of p38 were detected by RT-qPCR. b Venn diagram showing the miRNAs that target p38 in PicTar, TargetScan, and miRMap database. c Effects of laser irradiation on miRNAs that target p38 were detected by RT-qPCR. d Sequence alignment of the miR-24-3p base-pairing site in the 3′UTR of p38 mRNAs. e, f The targeting relationship of miR-24-3p and p38 was verified by dual-luciferase reporter gene and Western blotting. Data were expressed as mean ± SD. Comparison with control group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 2

The effect of miR-24-3p on the skin barrier in mice after 1064-QSNYL irradiation. a RT-qPCR detected the expression level of miR-24-3p in each group. b, c, f Multi-probe skin test system detected moisture content, skin elasticity, and TEWL immediately and up to the 28th day. d, e Hydroxyproline and SOD assays detected hydroxyproline and SOD content, respectively, up to 28 days. Data were expressed as mean ± SD. Comparison with NC group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Comparison with AD-miR-24-3p + irradiated group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001

Fig. 3

The effect of miR-24-3p on the skin barrier in mice after 1064-QSNYL irradiation. a RT-qPCR detected the expression level of K10, filaggrin, and AQP4. b Localization and quantification of K10, filaggrin, and AQP4 were performed by immunohistochemistry. Original magnification: × 40. Data were expressed as mean ± SD. Comparison with NC group, ^**P < 0.01. Comparison with AD-miR-24-3p + irradiated group, ^##P < 0.01

Fig. 4

The effect of miR-24-3p on collagen synthesis in mice after 1064-QSNYL irradiation. a, b Protein and mRNA expression of collagen I, collagen III, and collagen IV were detected by Western blotting and RT-qPCR, respectively. Data were expressed as mean ± SD. Compared with NC group, ^**P < 0.01, ^***P < 0.001. Comparison with AD-miR-24-3p + irradiated group, ^##P < 0.01, ^###P < 0.001

Fig. 5

The effect of miR-24-3p on collagen synthesis and skin barrier–related protein expression in HaCaT cells after 1064-QSNYL irradiation. a RT-qPCR detected the expression level of miR-24-3p in each group. b, c Protein and mRNA expression of collagen I, collagen III, and collagen IV were detected by Western blotting and RT-qPCR, respectively. d RT-qPCR detected the mRNA expression level of K10, filaggrin, and AQP4 in each group. e, f Localization and quantification of K10, filaggrin, and AQP4 by Immunofluorescence. Original magnification: × 40. Data were expressed as mean ± SD. Comparison with the control group, ^**P < 0.01, ^***P < 0.001. Comparison with mimic-NC + irradiated group, ^##P < 0.01, ^###P < 0.001