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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

Author: PanMin
Tutor: LuoDan
School: Nanjing Medical University
Course: Dermatology and Venereology
Keywords: Ultraviolet B (UVB) irradiation UVA (UVA) irradiation To photodamage light protection EGCG Fibroblasts (FB) Matrix metalloproteinase- 1 (MMP-1) Mitogen-activated protein kinases (MAPKs) c-Jun N -terminal kinase (JNK) RT-PCR Western blotting
CLC: R285
Type: Master's thesis
Year: 2008
Downloads: 169
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Abstract

Background: Ultraviolet (UV) radiation in sunlight is a very significant environmental factor, it is the main role in a person's skin. Size according to wavelength ultraviolet sunlight can be divided into three categories: 1) long-wave ultraviolet light (UVA), a wavelength of 320 ~ 400 mm, and a long-wave ultraviolet light, and almost all of it reaches the ground, can penetrate the dermis; 2) ultraviolet ( UVB), a wavelength of 280 ~ 320nm, can penetrate the skin, most applications can reach the ground; 3) short-wave UV (UVC), a wavelength of 200 to 280 nm are absorbed by the ozone layer and does not reach the ground. UVA major role in the dermis, its depth can reach the dermis Central fibroblast is the main target site of its role UVB part of the superficial dermal fibroblast cells is one of the target site. UVA and UVB in sunlight except skin cancer initiation and promotion, it is more important to UV radiation can easily promote skin aging process. Dermal collagen structure change as an important mechanism of photoaging. Photoaging performance in the dermis for increased degradation of dermal collagen, elastic fiber degeneration, resulting in wrinkles and skin elasticity decreases. Matrix metalloproteinases (MMPs) is the degradation of the extracellular matrix (ECM) is an important class of proteolytic enzymes, by hydrolysis, destruction and restructuring of ECM, eventually leading to skin connective tissue structure reconstruction, skin aging, clinical manifestations. Mitogen-activated protein kinase (mitogen-activated protein kinase, MAPKs) is a sequence of extreme conservative threonine and serine phosphorylation of enzymes in double-digit variety of oxidation, ultraviolet light, neurotransmitters and hormones stress-mediated factors, extracellular signal transfer to the nucleus, by fine-tuning the regulation of gene transcription, protein translation and metabolism, is one of the intracellular signal transduction system. In recent years found that the c-Jun N-terminal kinase (JNK) is one of the key elements of the MAPK pathway, involved in the signal transduction process of skin photoaging. JNK and p38 phosphorylation eventually start transcription factor activation protein (AP) 1. AP1 MMPs transcription necessary MMPs, thus contributing to high expression. By specific inhibition of JNK phosphorylation, to inhibit MAPKs signal pathway is involved in the process of photoaging, inflammation, apoptosis and proliferation. Epigallocatechin gallate catechin gallic acid (epigallocatechingallate, EGCG) is the main active ingredient of tea polyphenols (50%), anti-mutagenic, anti-oxidation, anti-viral, immune function, and induce tumor cell apoptosis role. Domestic and international studies have confirmed that green tea extract active ingredient EGCG can inhibit the UV radiation causes skin edema, relaxation and thickened with a light protective effect. The purpose of this project is to study and comparison of UVB and UVA irradiation on the expression of MMP-1 mRNA and protein levels in cultured the state human skin fibroblasts (FB) JNK-specific inhibitor SP600125 positive reference to explore EGCG light protection The mechanism of action and the correlation of the MAPK signaling pathway, whereby for green tea and its extract as a natural sunscreen applied to UVA and UVB wavelength ultraviolet protection and development to provide a theoretical proof and experimental data reference. Objective To observe the intervention effect of UVA and UVB irradiation on cultured the state human skin fibroblast cells (FB) the expression of MMP-1 mRNA and protein levels, as well as adding of EGCG or signal protein JNK specific inhibitor; observed UVB irradiation on cultured state servant skin fibroblasts phosphorylated JNK (p-JNK) protein expression and to join of EGCG or signal protein JNK specific inhibitor intervention role, whereby explore of EGCG inhibit UV-induced skin fibroblast MMP-1 secretion and its impact related regulatory molecules, can provide a theoretical basis for the development and practical application of natural sunscreen and experimental data reference. Method 1 cell culture: isolated Han youth circumcision postoperative skin fibroblast cells (FB), containing 10% fetal bovine serum in DMEM culture at 37 ° C in 5% CO ₂ conditions medium, adjusting the cell density of 1 × 10 ^{6 / ml, respectively quantitative depending on the purpose of the experiment were seeded in 6-well plates and 96-well plates, 80% of cell fusion when irradiated with ultraviolet B (UVB) . Ultraviolet radiation: quantitative experimental design timing UVB or UVA irradiation, drug intervention treatment before and after irradiation. 3 cell activity: MTT assay cell proliferation activity. 4.MMP-1 protein levels: ELISA assay of MMP-1 protein levels. The expression detected 5.MMP-1mRNA: RT-PCR assay of MMP-1mRNA expression. 6.JNK protein phosphorylation levels of detection: Western blotting assay protein phosphorylation of JNK (p-JNK). Results 1.EGCG or of SP600125 intervention of UVB or UVA irradiation-induced MMP-1 secretion levels. Pure EGCG or the SP600125 processing FB group between MMP-1 secretion was no difference (P> 0.05). Pure UVB or UVA irradiation group compared with unirradiated plus drug group, MMP-1 secretion increased significantly, the first two were the latter's 3.10-fold and 1.82-fold (P <0.05). Before and after UVB irradiation plus EGCG or SP600125 to reduce the secretion of MMP-1, MMP-1 secretion levels of the two groups of UVB radiation alone group to 61.8% and 48.9%, respectively (P <0.05). Similarly, before and after UVA irradiation plus EGCG or SP600125 also reduced secretion of MMP-1, MMP-1 secretion levels of the two groups were UVA irradiation alone group to 66.7% and 70.9% (P <0.05). 2.UVB or UVA timeliness of the FB MMP-1mRNA transcriptional expression. 12h after UVB irradiation FB and 24hMMP-1 mRNA expression significantly increased compared with normal control group (P <0.05), were 2.60 times and 2.66 times, the non-irradiated group. The UVA irradiation FB 24hMMP-1mRNA expression increased 1.98 times the non-irradiated group (P <0.05). 3.EGCG or SP600125 transcriptional expression levels of UVB or UVA irradiation induced MMP-1mRNA. UVB radiation alone group the MMP-1mRNA transcriptional expression than the non-illuminated three groups significantly increased UVB irradiation group was higher than that of the two groups of UVB EGCG/SP600125 processing are significant suppression of MMP-1mRNA transcription level of expression (P <0.05), the two groups of gray degrees were 71.9% and 40.4% of the radiation alone group. UVA irradiation alone group the MMP-1mRNA transcriptional expression compared to non-illuminated three groups significantly increased UVA irradiation group was higher than that of the two groups of UVA EGCG/SP600125 processing are significant suppression of MMP-1mRNA transcription level of expression (P <0.05), the two groups of gray degrees were 62.2% and 68.3% of the radiation alone group. The three groups of non-irradiated with UVA or UVB irradiation between the expression of MMP-1mRNA no difference (P> 0.05). P-JNK expression after 4.UVB irradiation FB timeliness and EGCG on UVB irradiation induced p-JNK protein expression levels. p-JNK protein 1/2h expression after UVB irradiation increased at 6h after irradiation started to decline. UVB radiation alone group, p-JNK expression compared with non-illuminated three groups significantly increased UVB radiation alone group and normal control group gray ratio of 1.68 the two groups of EGCG/SP600125 processing UVB than a simple the UVB irradiation set of p-JNK The level decreased expression (P <0.05), the gray level of the two groups were 27.9% and 32.4% of the UVB radiation alone group. UVB irradiation differences between the three groups of p-JNK protein expression was no significant difference (P> 0.05). Transcription and protein secretion of MMP-1mRNA conclusion UVB or UVA irradiation fibroblasts were significantly increased, and the trend is the same, but the amount of the efficiency and timeliness of the role of UVB light injury stronger than UVA. EGCG or SP600125 can inhibit UVB and UVA-induced MMP-1 protein secretion and mRNA transcription level. p-JNK protein 1/2h expression after UVB irradiation increased, decreased expression 6h after irradiation. EGCG or SP600125 inhibited UVB-induced p-JNK protein expression. Light protective effects of UVA or UVB irradiation FB EGCG may inhibit JNK participation MAPK signal transduction pathway, thereby inhibiting MMP-1 levels and activity.}

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

Abstract

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