The Effects Of EGCG Against UVA-Induced Photoaging In Skin Fibroblasts

Objective:Aging is a key issue in scientific research,and photoaging is a result of a number of complex mechanisms.EGCG(Epigallocatechingallate)is the main active ingredient in green tea polyphenols.EGCG has antioxidant effects on a variety of cells.EGCG may protect cells by activating antioxidant molecules and regulating the expression of related genes.In our previous research,we have found that baicalin can inhibit the related pathways of oxidative signaling and protect cells from aging damage.At the same time,the activation of reactive oxygen species(ROS)pathway is a key step leading to cell aging.Our study intends to explore the effects and molecular mechanisms of EGCG on UVA-induced human skin fibroblasts and provide theoretical support for the application of EGCG in the treatment of skin photoaging.Methods:1.Using CCK-8 method to detect cell proliferation activity to establish a suitable photoaging model and select the appropriate drug concentration of EGCG.2.Using β-galactosidase kit to detect the number and morphology of aging fibroblasts.3.Using real-time quantitative PCR to determine the length of telomeres,and use telomere repeat amplification and enzyme-linked immunosorbent assay to detect telomerase expression.4.Using ELISA kit to detect SOD,MDA,GSH-Px and CAT activities.5.Using flow cytometry to detect the cell cycle of aging fibroblasts.6.Using real-time quantitative PCR to detect the expression of MMP-1,TIMP-1,p66Shc,p53,p16 and c-myc mRNA levels.7.Using ELISA kit to detect the expression of TGF-β 1 in fibroblasts.Results:1.The CCK-8 detection method showed that we selected 10 J/cm2 as the appropriate dose for establishing a model of photoaging of fibroblasts,and selected EGCG concentration of 25 ug/ml as the subsequent experimental concentration.2.In the UNA group,we observed that among β-galactosidase-stained cells,positive cells showed a significant increase.Positive cells were significantly lower in the UVA+EGCG group than in the UVA group,and fewer positive cells were observed in the control and EGCG groups.3.Real-time quantitative PCR experiments showed that EGCG can lengthen the length of telomeres of fibroblasts,but cannot lengthen the length of telomeres of aging fibroblasts induced by UVA.UVA irradiation affect telomerase activity,while EGCG has no significant effect on telomerase activity of fibroblasts.4.ELISA experiments show that EGCG significantly increases the content of SOD,GSH-Px and CAT in aging fibroblasts,and inhibits the generation of MDA,which means that EGCG can increase the antioxidant capacity of aging fibroblasts induced by UVA and inhibit related oxidative damage.5.Flow cytometry detection of the cell cycle suggested that EGCG delayed the G1 phase of UVA-induced aging fibroblast.6.Real-time quantitative PCR results showed that EGCG significantly reduced the expression of MMP-1,p66shc,and c-myc mRNA in UVA-induced aging cells.MMP-1,p66shc,and c-myc are oxidative damage-related molecules,which means EGCG can reduce UVA damage to fibroblasts by inhibiting related oxidative damage.7.ELISA experiments show that EGCG can enhance the expression of TGF-β1 in UVA-induced aging fibroblasts,the enhanced of TGF-β1 activity can delay aging of fibroblasts.Conclusion:This experiment shows that EGCG can increase the expression of SOD,GSH-Px and CAT in aging fibroblasts and inhibit the expression of MDA,thereby improving the antioxidant capacity of aging cells.At the same time,EGCG can inhibit the expression of MMP-1,p66shc,c-myc to further inhibit the related oxidative damage pathway,EGCG also can delay cell aging by enhancing the expression of TGF-β1.Also provide strong evidence for the application of EGCG in the treatment of skin photoaging and skin cancer.