A Mechanism Mediated By Cyanidin-3-O-glucoside Protects Human Dermal Fibroblasts Against UVA-induced Damage

Objective:Ultraviolet A(UVA)can cause skin erythema,pigmentation,photoaging and skin carcinogenesis.Topical application or consumption of natural compounds can prevent ultraviolet damage to the skin.Autophagy is an important process in the selective degradation of cellular components,which occurs in all eukaryotes.As a monomer of anthocyanin with the ability of oxygen free radical absorption,there is little research on the effect of Cyanidin-3-o-glucoside(C3G)on the damage of human dermal fibroblasts induced by UVA.In this experiment,the model of light damage wasestablished by UVA irradiation on primary HDFs.The effect of C3G on the primary HDFs damage induced by UVA was studied,and the effect of autophagy on the anti-light damage of C3G was preliminarily discussed by the intervention of autophagy inhibitor,which provided the experimental basis for the prevention and treatment of primary HDFs light damage by natural compounds.Methods:1 Effect of UVA on apoptosis and autophagy level of primary HDFs.(1).Enzymatic digestion was used to extract primary HDFs from human foreskin tissue.(2)Experimental groups were divided into normal control group and different energies UVA groups.(3)3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)method was used to detect cell activity,western blot assay was used to express the level of apoptosis related protein Bcl-2,Bax and autophagy related protein Atg5,LC3 in primary HDFs.The autophagosome was observed by electron microscopy.2 Inhibitory effect of C3G on UVA-induced primary HDFs damage.(1)MTT assay was used to measure cell viability and determine the concentration of C3G.(2)Experimental groups were divided to blank control group,UVA injury group,C3G+UVA group and C3G group.(3)Immunofluorescence and flow cytometry was used to observed and detected the reactive oxygen species(ROS) expression level;cell apoptosis was detected by flow cytometry;the expression of apoptosis related proteins and autophagy related proteins of primary HDFs were detected by Western blot assay.3 Effect of autophagy inhibitor 3-MA on that C3G protects primary HDFs against UVA-induced damage.(1)MTT assay was used to measure cell viability and determine the concentration of 3-MA.(2)Experimental groups were divided to blank control group,UVA damage group,C3G+UVA group,UVA+C3G+3-MA group,C3G group and 3-MA group.(3)Immunofluorescence and flow cytometry were used to observed ROS expression,cell apoptosis was detected by flow cytometry and the expression of apoptosis related protein of primary HDFs were detected by western blot assay.Results:The experimental results show:1 The primary cells extracted from the foreskin tissue were expressed Vimentin.In the light damage model,with the increase of UVA energy,HDFs activity was decreased,apoptosis levels was gradually increased.Compared with the control group,there was statistical significance(P<0.05).The level of autophagy increased when UVA energy was 0-4J/cm~2,and then decreased with the increase of UVA energy.In addition,the optical damage model is that primary HDFs was irradiated with the energy of 12J/cm~2 UVA according to MTT experiment.2 In 12J/cm~2 UVA induced primary HDFs optical damage model,C3G can significantly reduce the content of ROS,increased the ratio of anti-apoptotic protein Bcl-2 to apoptosis protein Bax,decreased cleaved caspase-3/caspase-3 ratio,and increased autophagy protein Atg5 and LC3 levels(P<0.05). 3 The autophagy inhibitor 3-MA was used to intervene the photoprotective effect of C3G against 12J/cm~2 UVA-induced primary HDFs damage.ROS level and apoptotic level were significantly increased and the ratio of anti-apoptotic protein Bcl-2 to pro-apoptotic protein Bax was significantly decreased in the UVA+ C3G+3-MA treatment group compared with that C3G+UVA group(P<0.05).Conclusion:1 The cells extracted from foreskin tissue were the primary HDFs.UVA irradiation could significantly increase the apoptosis level of primary HDFs,and higher energy UVA could reduce the autophagy level of primary HDFs.12J/cm~2 UVA was chosen to irradiate the primary HDFs as the optical damage model.2 C3G can significantly reduce the apoptosis level and increase the autophagy level of primary HDFs in the optical damage model.3 The mechanism of C3G reducing the apoptosis of primary HDFs in the optical damage model may be related to induce the level of autophagy of HDFs.