Photoprotection Effects Of EGCG On Reducing Protein Secretion And MRNA Expression Of MMP-1 In Human Skin Fibroblasts After UVB Or UVA Irradiation And The Relevant Molecules Mechanisms

BackgroundUltraviolet irradiation of sunlight is a significant environmental factor,which can be divided into UVA,UVB and UVC according to wavelength.The wavelength of UVA is 320-400nm,which can penetrate to dermis.The wavelength of UVB is 280-320nm,which can penetrate to several millimeter of skin.UVA and most of UVB can reach to the earth surface and human skin,but most of UVC can not reach the skin because of absorption by the ozonosphere.Fibroblasts in dermis are the major target cells of UVA irradiation and also the targets of UVB,part of which can reach to superficial layer of dermis.Ultraviolet irradiation including UVA and UVB is not only the switch and promotion of skin tumors,but also can lead to skin photoaging. Dermal collagen structural change is one of the important mechanism of photoaging.Photoaging presents as increasing degradation of dermis collagen and degeneration of elastic fibers,which develop wrinkles and elasticity declining.Matrix metalloproteinases(MMPs)are a group of important proteolysis enzymes to degrade extracellular matrix(ECM)and they can hydrolyze and breakdown ECM,lead to rearrangement of connective tissues and clinical features of photoaging.Mitogen-activated protein kinases(MAPKs)are a kind of extremely conservative double phosphorylational threonine and serin enzyme system,which can transfer the extracellular signal into cellular nucleus under oxidation and ultraviolet.MAPKs can control transcription of genes,translation of proteins and metabolism,so MAPKs are mainly signal transduction pathways of cells,c-Jun amino terminus kinase(JNK) is a key molecule of MAPKs and it is often involved in the signal transduction of photoaging.JNK and p38 switch AP1 after phosphorylation.AP1 is essential factor to transcription of MMPs because AP1 can increase the production of MMPs.We can inhibit the process of photoaging,inflammatory reaction,apoptosis by interfering the MAPKs signal transduction pathway,such as specifically inhibiting phosphorylation of JNK and / or p38.Epigallocatechingallate(EGCG)is one kind of major and most effective component of TP(tea polyphenols)from green tea.It has been proved worldwide that EGCG is able to improve antioxidation, anti-inflammation and anti-carcinogenesis.Some researches have confirmed EGCG has photo-protection effects by inhibition of edema, looseness and thickness induced by UVR.The study aimed to compare the different expressions of MMP-1 protein and mRNA after UVA or UVB irradiation in skin fibroblasts and investigate the photo-protection of EGCG.on UVA or UVB induced MMP-1 expressions.The experiment was then expended to explore the photoprotective mechanism of EGCG on MAPKs signal transduction pathway.The results will contribute to the development and application of the natural sunscreens from TCM.ObjectiveTo compare the different expressions of MMP-1 protein and mRNA in fibroblasts after UVA and UVB irradiation and investigate the photo-protection of EGCG.on UVA and UVB induced MMP-1 expressions.The experiment was then expended to explore the photoprotective mechanism of EGCG on MAPKs signal transduction pathway.The results will contribute to the development and application of the natural sunscreens from TCMMaterials and methods1.Cell culture:primary fibroblasts(FB)were isolated from healthy human skin samples and cultured in DMEM medium with 10%calf serum;The above cells were plated in 96 wells and 6 wells with an equal number of cells(10~6 cells).2.Ultraviolet irradiation:Fibroblasts were shammed or irradiated according to the designed dose and EGCG or JNK inhibitor was added into the cultures after UV radiation.3.Cell viability assay:MTT assay was used to record cell proliferation and cellular viability.4.Determination of MMP-1 level:The secretion level of MMP-1 was detected with ELISA Kits.5.Determination of MMP-1 mRNA expression:The expression of MMP-1 mRNA was detected by RT-PCR.6.Determination of p-JNK protein expression:The expression of p-JNK protein was measured by Western blotting.Results1.Effects of EGCG or JNK inhibitor on the UVB or UVA induced expression of MMP-1 protein level in human skin fibroblasts.Compaed with the sham group,the MMP-1 levels protein in UVB or UVA irradiated groups were increased 3.10 and 1.82 times, respectively(P＜0.05).The UVB induced MMP- 1 secretion level was decreased to 61.8%and 48.9%after the cultures were treated by EGCG or JNK inhibitor(P＜0.05).The UVA induced expression of MMP-1 was decreased to 66.7%and 70.9%after the cultures were treated by EGCG or JNK inhibitor(P＜0.05).2.Time-effects of MMP-1mRNA expression after UVB or UVA irradiation in human skin fibroblasts.The expressions of MMP-1mRNA increased significantly 12h(2.60 folds)and 24h(2.66 folds)after 30mJ/cm~2 UVB irradiation,compared with the sham group(P＜0.05).The expression of MMP-1mRNA increased significantly 24h after 10J/cm~2UVA irradiation,which was 1.98 times higher than that of the sham group(P＜0.05).3.Effects of EGCG or JNK inhibitor on the UVB or UVA induced expression of MMP-1 mRNA in human skin fibroblasts.The expression of MMP-1mRNA irradiated by UVB increased significantly than the sham group.The UVB induced expression of MMP-1 mRNA was down regulated to 71.9%and 40.4%by EGCG or JNK inhibitor treatment.The expression of MMP-1 mRNA irradiated by UVA increased significantly than that of the sham group. The expression of MMP-1 mRNA of two group treated by EGCG or JNK inhibitor after UVA irradiation decreased to compared with pure UVA irradiation group.The UVB induced expression of MMP-1mRNA was decreased to 62.2%and 68.3%after the cultures were treated by EGCG or JNK inhibitor.4.Effects of EGCG or JNK inhibitor on the UVB induced expression of p-JNK in human skin fibroblasts.The expression of p-JNK increased significantly 0.5h and decreased 8h after UVB irradiation,.The expression of p-JNK protein irradiated by UVB increased 1.68 times compared to the control group.The UVB induced expression of p-JNK was decreased to 27.9%and 32.4% after the cultures were treated by EGCG or JNK inhibitor.Conclusions UVB or UVA irradiation can increase the MMP-1 protein and mRNA level in human skin fibroblasts significantly.So far as the potency of UVR to the cultures fibroblasts is concerned,UVB and UVA have the similar efficiency on the MMP-1 secretion level,but the effect of UVB seems stronger than that of UVA under the designed dose and and time condition.EGCG or JNK inhibitor can decrease the MMP-1 protein and mRNA level increased by UVB or UVA irradiation.The fact that the expression ofp-JNK involved in MAPK signal transduction pathway was modulted by EGCG.may be one part of the mechanisms related to the photo-protection effects of EGCG.