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The Effect of Daidzein on Collagen Synthesis and the Possible Mechanism

Author: WengYuJing
Tutor: YeXiYun
School: East China Normal University
Course: Biomedical
Keywords: compounds screening collagen synthesis daidzein human dermalfibroblasts BALB/C mouse
CLC: R285
Type: Master's thesis
Year: 2010
Downloads: 37
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Abstract

Collagen is the primary component of the skin dermis, which is known to perform a pivotal function in the maintenance of the structure of the skin dermis. Ageing of the skin is primarily related to reductions in the levels of collagen. Both the quantity and quality of collagen are primarily related to skin ageing. In this study, we aim at selecting a possible anti-wrinkle agent and detecting its effect on collagen production in vitro and in vivo.The research can be divided into three parts:1、The construction of recombinant plasmid and compounds screening We amplified the regulatory element of human a2(I) collagen gene(COL1A2) from the total DNA, and inserted the sequence into PGL-3plasmid, then transiently transfected the plasmid into293T cells by liposome. After treatment of different compounds in a suitable concentration, the amounts of luciferases in cells were measured. Thus, the compounds which could induce the luciferase production can activate the expression of COL1A2either. In this way, we establish a stable model for screening compounds which can activate the activity of COL1A2promoter. Then using this model, we select daidzein as a possible anti-wrinkle agent.2、The effects of daidzein on collagen synthesis in vitro and the possible mechanism After24h or48h treatment of0.5μg/ml,5μg/ml,50μg/ml daidzein respectively, the human dermal fibroblasts we detected the changes of those features: the change of cell cycle by flow cytometry; the change of mRNA levels of matrix metalloproteinase-1(MMP-1), matrix metalloproteinase-2(MMP-2) and tissue inhibitor of metalloproteinase-1(TIMP-1) by RT-PCR; the change of protein levels of type I collagen, type III collagen, MMP-1and MMP-2by Elisa essay. The results suggest:daidzein has no effect on fibroblasts proliferation; daidzein can improve the mRNA level and protein level of type I collagen and type III collagen dose dependently (P<0.05and P<0.01); daidzein can decrease the amount of MMP-1and MMP-2but decrease the transcription of TIMP-1(P<0.05and P<0.01) dose dependently.3、The effects of daidzein on collagen synthesis in vivo and the possible mechanism Daidzein was dissolved by water containing30%glycerol in a concentration of200μg/ml, then100μl of this solution was provided to the right part of the dorsal skin of BALB/c mouse, and the left part was provided with100μl30%glycerol as control. After six weeks of treatment, the skin elasticity and hydration were measured, then the two parts of the skin were used to detect the mRNA levels of type I collagen, type III collagen, MMP-2, tissue inhibitor of metalloproteinase-2(TIMP-2) and transforming growth factor-β (TGF-β) by RT-PCR, and the amount of type I collagen and type III collagen by Immunohistochemistry. The results show that: daidzein improves the skin elasticity and hydration significantly(P<0.05and P<0.01); the transcription levels of type I collagen and type III collagen are significantly increased by daidzein (P<0.01) and the amount of them in skin is increased obviously. Daidzein can significantly activates the TGF-P transcription.In conclusion, the data acquired in this study demonstrate that:1、To insert the regulatory element of COL1A1into PGL-3plasmid and then transfect the293T cell, we can establish a model for screening compounds which can induce the transcription of type I collagen.2、Daidzein increase the synthesis of type I collagen and type III collagen in human dermal fibroblasts, and can reduce the degradation of collagen by inhibiting the synthesis of MMP1and MMP2and activating the TIMP2transcription.3、Daidzein increase the amount of type I collagen and type III collagen in the skin of BALB/C mouse by activating the transcription of them and in thus improves the skin elasticity and hydration. Daidzein possibly improves the synthesis of collagen by inducing the transcription of TGF-β.Therefore, based on these results, we suggest the possibility that daidzein may be considered as possible wrinkle-reducing candidates for topical application.

The Effect of Daidzein on Collagen Synthesis and the Possible Mechanism

Abstract

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