Mechanisms of Dynasore induced procollagen type I in wounded skin fibroblast cells CCD-966SK and in vivo ICR mice

• Main Authors: Siang-Rong Lu, 呂享融
• Other Authors: 鄭幼文
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/42383844224072320689

碩士 === 臺北醫學大學 === 藥學研究所 === 98 === Collagen is the most abundant protein in humans, it exists in the connective tissue, and with high tension and tensile strength.The collagen molecules assemble into functional integrity of tissue such as skin, ligament, tendon, bone and cartilage which is the main component of the extracellular matrix especially in human skin. Collagen not only maintains the elasticity of skin and muscle but also plays a promoting role during the process of wound healing. Tissue repair has been divided into two phases, one is inflammatory phase, a proliferation phase with synthesis of new connective tissue and epithelial wound closure, the other is maturation phase once the epidermal barrier has been re-established. The most important step of wounding healing is the predominant form of collagen in the human skin and is produced mainly by dermal fibroblasts. Dynasore, a small organic molecule is a newly discovered cell-permeable dynamin inhibitor, blocks the formation of clathrin-coated vesicles through its inhibitory effects on the GTPase activity of dynamin. Studies suggested that Dynasore enhances TGF-β1–induced plasminogen activator inhibitor-1 expression, and might be through JNK signal pathway in human MeT-5A cells. More and more increasing evidences support not only the Smad pathway but also others such as mitogen-activated MAPK and PI3K pathways play the important role in TGF-β signaling. In addition, type I collagen produce relating to TGF-β, PI3K pathway has reperted. In this study, we investigated the effects of Dynasore on human skin fibroblast of wound healing in vitro and in vivo. Results of MTT assay showed that no adverse effect of Dynasore at the concentration up to 100 μM when treated fibroblast. We found Dynasore concentration-dependent increased procollagen type I induction cell proliferation and migration. These effect might be due to induce ERK1/2 and MMP-1 phosphorylation. Recently, TGF-β/Smad3 pathway is recognized in controlling the wound healing, and PI3K also related. Our data suggested Dynasore induced Smad3, Akt, mTOR phosphorylation as well as procollagen type I expression. When treated with ERK, Akt and mTOR inhibitors PD98059, LY294002 and rapamycin, respectively. We found that only LY294002 can inhibit Dynasore -induced procollagen type I protein expression. We conclued that Dynasore -induced procollagen type I protein expression might be through PI3K/Akt pathway. Although PD98059 inhibited the ERK1/2 phosphorylation and MMP-1 expression, but it cannot suppress procollagen type I. indicated that the formation of procollagen type I is indepented to ERK/MAPK pathway and MMP-1. In the in vivo study, ICR mice were created a 6 mm diameter wound on the back by skin biopsy punch. Topical treating with Dynasore, bFGF(basic fibroblast growth factor) as positive control for 14 days, then recorded the wound area every day. Our results showed 3 mg/mL Dynasore significantly promoted wound healing better than bFGF after Day 5. In the mucositis animal model, 0.3 mg/mL Dynasore shown a better outcome than control group. Basic fibroblast growth factor(bFGF) regulates proliferation, differentiation, migration and apoptosis. BFGF has the potential to accelerate wound healing and improve the quality of wound healing by regulating the balance of ECM synthesis and degradation. In this study, Dynasore has the equivalent role of bFGF, is also a non-protein compound, and stable when storage.We suggest that the effectiveness of Dynasore including non-toxic in high dose, excellext effect and easy to preserve. It might be a potential drug candidant in the future. The purpose of this study is to find out a non-protein compound to replace the use of bFGF on wound healing and anti-mucositis, we also wish can develop a good and potential chemical for medical purpose in the drug development.