Kan Sun1, Wei Wang1, Chuan Wang1, Guojuan Lao1, Dan Liu1, Lifang Mai1, Li Yan1, Chuan Yang2, Meng Ren3. 1. Department of Endocrinology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510120, PR China. 2. Department of Endocrinology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510120, PR China. Electronic address: 13640242307@139.com. 3. Department of Endocrinology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510120, PR China. Electronic address: renmeng80@139.com.
Abstract
OBJECTIVE: Accumulation of advanced glycation end products (AGEs) contributes to the development of diabetic ulcers. Recent evidence indicates that AGEs administration enhanced autophagy in many cell types. As a positive trigger of autophagy, the effect of AGEs on autophagy in skin tissues and fibroblasts remains unknown. METHODS: Skin tissues were isolated from Spreqne-Dawley rats and immunohistochemical staining was performed to analyze the location of LC3 and FOXO1 in skin tissues. Then primary cultured foreskin fibroblast cells with treated with AGEs and the effect of AGEs on autophagy was investigated. Protein level expressions of LC3, Beclin-1 and FOXO1 in fibroblasts were analyzed by Western blotting. Autophagic flux is detected with autophagy inhibitor chloroquine and mRFP-GFP-LC3 tandem construct. RESULTS: Compared with skin from normal rats, immunohistochemical staining shows a predominant LC3 localization in fibroblasts cytoplasm in diabetic rats. Elevated expression of FOXO1 also existed in diabetic rats dermis fibroblasts when compared with normal rats in immunohistochemical analysis. In human skin fibroblasts cells, AGEs administration stimulated the autophagy related LC3-II/LC3-I and Beclin-1 expressions and increased autophagy flux. In mRFP-GFP-LC3 puncta formation assays, both autolysosome and autophagosome were increased in human fibroblasts after treatment with AGEs. Fibroblasts exposed to AGEs also have increased FOXO1 expression compared with control group. CONCLUSION: AGEs could induce autophagy at least in part via regulating the FOXO1 activity in diabetic skin tissues and fibroblasts.
OBJECTIVE: Accumulation of advanced glycation end products (AGEs) contributes to the development of diabetic ulcers. Recent evidence indicates that AGEs administration enhanced autophagy in many cell types. As a positive trigger of autophagy, the effect of AGEs on autophagy in skin tissues and fibroblasts remains unknown. METHODS: Skin tissues were isolated from Spreqne-Dawley rats and immunohistochemical staining was performed to analyze the location of LC3 and FOXO1 in skin tissues. Then primary cultured foreskin fibroblast cells with treated with AGEs and the effect of AGEs on autophagy was investigated. Protein level expressions of LC3, Beclin-1 and FOXO1 in fibroblasts were analyzed by Western blotting. Autophagic flux is detected with autophagy inhibitor chloroquine and mRFP-GFP-LC3 tandem construct. RESULTS: Compared with skin from normal rats, immunohistochemical staining shows a predominant LC3 localization in fibroblasts cytoplasm in diabeticrats. Elevated expression of FOXO1 also existed in diabeticrats dermis fibroblasts when compared with normal rats in immunohistochemical analysis. In human skin fibroblasts cells, AGEs administration stimulated the autophagy related LC3-II/LC3-I and Beclin-1 expressions and increased autophagy flux. In mRFP-GFP-LC3 puncta formation assays, both autolysosome and autophagosome were increased in human fibroblasts after treatment with AGEs. Fibroblasts exposed to AGEs also have increased FOXO1 expression compared with control group. CONCLUSION: AGEs could induce autophagy at least in part via regulating the FOXO1 activity in diabetic skin tissues and fibroblasts.