Screening System of Blocking Agents of the Receptor for Advanced Glycation Endproducts in Cells Using Fluorescence
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概要
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Activation of the receptor for advanced glycation endproducts (RAGE) triggers cellular responses implicated in the pathogenesis of diabetic complications; blockade of RAGE has been shown to inhibit the development of diabetic complications. To develop a screening system to identify novel disruptors of advanced glycation endproducts (AGE)-RAGE binding, we used an AGE-RAGE binding system in RAGE-overexpressing cells; test compounds were screened using this system. To construct human RAGE-overexpressing cells, mouse mesangial cells (MMCs) were stably transfected with the pcDNA-human RAGE (hRAGE) vector and selected under 1 mg/mL gentamicin (G418). RAGE expression in hRAGE-overexpressing MMCs was analyzed by Western blotting with specific RAGE antibody. To identify novel disruptors of AGE-RAGE binding, 50 single compounds and AGE-bovine serum albumin (BSA)-Alexa 488 (AGE-BSA labeled with Alexa 488) were treated to the hRAGE-overexpressing MMCs. Nonbinding AGE-BSA-Alexa 488 was washed and fluorescence measured by microtiter plate reader (excitation wavelength, 485 nm; emission wavelength, 528 nm). In hRAGE-overexpressing cells, only treatment with AGE-BSA-Alexa 488 significantly increased fluorescence intensity in a dose-dependent manner. Of 50 compounds tested, genistein disrupted AGE-RAGE binding in a dose-dependent manner. This AGE-RAGE binding system using AGE-BSA-Alexa 488 in hRAGE-overexpressing cells was suitable for screening of agents that disrupt AGE-hRAGE binding.
- 公益社団法人 日本薬学会の論文
著者
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Kim Young
Korea And College Of Pharmacy Chungbuk National University
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Kim Jin
Korea Advanced Inst. Sci. And Technol. Daejeon Kor
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Jung Dong
Korean Medicine-Based Herbal Drug Research Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM)
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Kim Jin
Korean Medicine-Based Herbal Drug Research Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM)
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Kim Young
Korean Medicine-Based Herbal Drug Research Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM)
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