The Effects Of Blue Light On Skin Aging And Its Molecular Mechanism

Objective:Previous studies have shown that blue light may be involved in the occurrence of skin photoaging.This study aims to explore the phenotypic characteristics and molecular mechanism of blue light-induced skin aging,providing a theoretical basis for the precise protection of photodermatosis.Methods:In this study,the SFIM-300 spectral scintillation illuminance meter was used to test the irradiation intensity of common light sources in daily life to clarify the specific distribution and dose of blue light in our living environment.The human fibroblast cell line(HFF-1)and human primary skin fibroblasts were used for cell experiments.The RTCA system of Roche was used to detect the effect of blue light on HFF-1 cells and determine the blue light dose used in the subsequent experiments.Use R language to run Normfinder code to analyze the relative stability of housekeeping genes and to screen the internal reference available for subsequent research.Three light sources of 420nm,450nm blue light and 650nm red light were selected,and four blue light dose gradients of0 J/cm~2,10 J/cm~2,15 J/cm~2,and 20 J/cm~2 were used to irradiate HFF-1 and primary cells.The study selected C57BL/6 mice and BALB/c nude mice to perform blue light irradiation experiments to observe whether blue light can cause damage to mouse skin,and pathological examination of mouse skin.2.In this study,the RNA-Seq detection method was used to analyze the differential genes before and after the blue light irradiation of HFF-1 cells,and the GO gene enrichment analysis and differential gene pathway enrichment analysis.3.In this study,the reaction oxygen species detection kit was used to detect the change of ROS expression level after HFF-1 cells were irradiated with blue light,and the cells were treated with ROS inhibitors to analyze whether ROS was one of the reasons for blue light-induced cell phenotype.Western Blot method was used to detect the phosphorylated expression of Smad2/3 protein,and to detect TGFBR2protein and Smad7 protein.TGF-?cytokines were used to stimulate cells and overexpress TGFBR2 to determine whether blue light regulated the expression of type ? collagen through the TGF-?signaling pathway.The expression of JNK phosphorylated protein and downstream signaling molecule AP-1/c-Jun phosphorylated protein was detected by western blot method.HFF-1 cells were treated with SP600125,an inhibitor of the JNK pathway,and the effect of the pathway on the cells was examined.Western blot method was used to detect the protein phosphorylation changes of epidermal growth factor receptor(EGFR)and p70S6K.Gefitinib(EGFR pathway inhibitor),MHY1485(p70S6K protein phosphorylation activator)and rapamycin(p70S6K protein phosphorylation inhibitor)were used to treat HFF-1 cells,and the phenotype changes were observed.Results:1.The blue light radiance in sunlight is the highest,and there is also a certain radiance in the blue light in all kinds of light sources in daily life.Blue light generally exists in natural and artificial light sources and is inseparable from daily life,so the potential adverse effects of blue light exposure on our skin cannot be ignored.RTCA system detected the effect of blue light on HFF-1 cells.The results showed that 26 J/cm~2and higher radiation doses had toxic effects on fibroblasts,13 J/cm~2 and lower radiation doses had no toxic effects on cells.The reference gene SDHA gene expression is relatively stable in HFF-1 and primary cells irradiated with blue light,and is the optimal reference gene.After blue light irradiation,the expressions of COL1A1,COL1A2,and COL3A1 genes of HFF-1 and primary cells decreased,while the expressions of MMP1,MMP3,and IL-1?genes increased,and all showed a dose-dependent increase.20 J/cm~2blue light irradiation dose can induce the increased expression of MMP1 protein in HFF-1 and primary cells,and inhibit the expression of COL1 protein.Two wavelengths of blue light have the same damage effect on cells.Red light irradiation does not cause changes in the expression of skin aging-related genes,and when irradiating cells with the same dose as blue light,it does not cause changes in cellular protein expression.after the blue light irradiation,the skin of the back of C57BL/6 mice and BALB/c nude mice showed rough scaliness,wrinkles,skin thickening and loss of elasticity.HE staining of mouse skin showed that the skin layer of the mice in the light group showed irregular thickening,the thickness of the true and epidermis was significantly higher than that of the control group,and the tissue structure in the dermis was disordered.Masson's staining showed that the collagen content of the dermis layer of the mice in the light group was reduced,the collagen fibers were degenerated,arranged disorderly,and the thickness and uneven distribution.The immunohistochemical staining of MMP13 in the skin of irradiated mice significantly increased the number of MMP13 positive cells,and the expression of MMP13 gene increased significantly.2.Differential gene analysis of HFF-1 cells before and after blue light irradiation shows that the up-regulated genes are mainly distributed on genes related to stress response activation,which is related to the stress response of cells caused by blue light irradiation.The down-regulated genes focus on chromatin synthesis and cell cycle,which means that blue light irradiation affects cell division and cycle by inhibiting chromatin and DNA synthesis,thereby inhibiting cell proliferation.In the RNA-Seq data,eight genes were screened according to the screening principle to verify the differentially expressed gene levels.The results showed that the expression of these genes increased or decreased significantly after blue light irradiation,and had blue light dose dependence.3.The ROS expression level of HFF-1 cells increased significantly after blue light irradiation.When the cells were treated with ROS inhibitors,the ROS expression after blue light irradiation was inhibited,and the phenotypic changes induced by blue light were partially inhibited.Blue light inhibits the phosphorylation of downstream Smad 2/3 protein by inhibiting TGFBR2 protein,thereby inhibiting the collagen synthesis of HFF-1 cells,and further inhibiting the TGF-?signaling pathway by promoting the expression of Smad7.After stimulation with TGF-?factor,the expression of TGFBR2 protein increased in cells without blue light irradiation,and the expression of COL1A1 gene and protein increased significantly,while the expression of TGFBR2 and COL1A1 after TGF-?factor stimulation in cells after blue light irradiation did not increase.Overexpression of TGFBR2 cells can reverse the type ? collagen expression of HFF-1 cells suppressed by blue light.The phosphorylation level of JNK protein and c-Jun protein were activated 1 hour after blue light irradiation.Treatment of HFF-1 cells with SP600125,an inhibitor of the JNK pathway,showed that SP600125 can promote the expression of type ? collagen in HFF-1 cells and inhibit the expression of SMAD7 and MMP1,thereby reversing the blue light-induced changes in cell phenotype.The EGFR protein undergoes significant phosphorylation changes 1 hour after blue light irradiation,and gefitinib can significantly inhibit blue light-induced phosphorylation of EGFR protein,thereby reducing the expression of MMP1 in cells.Gefitinib can also significantly activate the phosphorylation level of p70S6K protein,and further restore the phosphorylation level of p70S6K protein that is inhibited by blue light.MHY1485 can inhibit the expression of MMP1 in HFF-1 cells,and inhibit the expression of MMP1 induced by blue light,and reduce the expression level of MMP1 after blue light irradiation.On the other hand,inhibition of p70S6K protein phosphorylation can induce MMP1 expression in HFF-1 cells,and further promote blue light-induced MMP1expression.Conclusion:1.Blue light is ubiquitous in life and has a cumulative effect on the skin.Blue light can induce the occurrence of skin photoaging,and this effect is wavelength-specific in visible light.2.The genes up-regulated after blue light irradiation on HFF-1 cells were mainly distributed on genes related to stress activation,and the down-regulated genes were concentrated on chromatin synthesis and cell cycle.3.Blue light induced skin aging in a ROS-independent manner,and might induce skin aging through multiple molecular pathways(TGF-?pathway,JNK pathway,and EGFR pathway).Blue light inhibited the synthesis of type ? collagen(COL1)by inhibiting the TGFBR2/Smad2/3 pathway and activating the JNK/AP-1/Smad7 pathway.Blue light promoted the synthesis of matrix metalloproteinase 1(MMP1)by activating the JNK/AP-1 pathway and the EGFR/p70S6K pathway.