抑制瘢痕形成的ACE相关活性肽/抑制剂的筛选与作用机制研究

Wound scarring is always a major focus in the field of plastic surgery due to its difficulty in clinical treatment. However, the molecular mechanism of scar formation is not well understood, and the curative effect is not well achieved. In recent years, it has been recognized that the local renin-angiotensin system (RAS) participates in the fibrosis of multiple organs via promoting cellular proliferation and synthesis of extracellular matrix. However, these studies of RAS in scar were not systematic, only focused mainly on the effects of angiotensin converting enzyme (ACE) and angiotensin II (Ang II), and the mechanisms are not very clear; whether the various peptides/inhibitors regulated by ACE can be used as the effective ingredients of drugs or applied topically to prevent scar formation has not been recognized, and we also do not known which one is the best. We have verified that ACE was involved in scar formation when the previous National Natural Science Foundation (No. 81372072) was performing; and we recognized that some of the ACE related peptides and their inhibitors affected the scar formation via TGF-β pathway. Based on these facts, we propose here to screen these peptides/inhibitors systematically, and investigate how they are involved in wound healing and scar formation. To the potential peptides related to fibrosis in the literatures (Ang II、SP、AcSDKP、Ang 1-7 and Bradykinin) and their inhibitors (including the antagonists for Ang II、SP and Bradykinin, and the inhibitors for AcSDKP, Ang 1-7 and ACE), an in vitro analysis will be performed using the mouse fibroblast cells (NIH3T3) and fibroblasts expressing no ACE (ACE knockout) or excessive ACE (transgene); then, drugs given to the whole body, topical application and transdermal drug delivery mediated by CO2 fractional laser using the mice and rats will be performed step by step; so the best one will be screened out after the 4 steps, and the mechanisms of action will be found. The completion of this proposal will not only extend our knowledge and understanding about wound healing and scar formation, but ultimately, may provide theory evidences for new drugs to inhibit scar formation aiming at ACE.

瘢痕研究一直是整形外科界的焦点之一，但其形成机制尚不明确，疗效仍不理想。近年发现肾素-血管紧张素系统（RAS）参与多种器官纤维化；但目前仅有RAS与瘢痕相关性的零散研究，且主要局限于血管紧张素转换酶（ACE）和血管紧张素II的效应，ACE调控的多种活性肽及对应抑制剂能否用作抗瘢痕药物的有效成分、能否局部外用、何者最有效均不明确。我们前期证实了ACE对瘢痕的影响，并发现部分活性肽和抑制剂通过TGF-β调控瘢痕形成。本项目拟作系统的筛选研究，并揭示其调控模式和机制。对文献提示可能影响纤维化的ACE相关活性肽（缓激肽、AcSDKP等）及抑制剂，通过体外分析（小鼠成纤维细胞系、ACE 基因敲除和过表达细胞）、全身给药、局部给药、点阵激光介导的靶向透皮给药（小鼠、大鼠）4层筛选，逐步筛选出高效成分，明确作用机制。该研究将拓展对瘢痕形成机制和治疗的认识，为下一步制备靶向ACE的抗瘢痕药物提供理论依据。

瘢痕研究一直是整形外科界的焦点之一，但其形成机制尚不明确，疗效仍不理想。近年发现肾素-血管紧张素系统（RAS）参与多种器官纤维化；但目前仅有RAS与瘢痕相关性的零散研究，且主要局限于血管紧张素转换酶（ACE）和血管紧张素II的效应，ACE调控的多种活性肽及对应抑制剂能否用作抗瘢痕药物的有效成分、能否局部外用、何者最有效均不明确。我们前期证实了ACE对瘢痕的影响，并发现部分活性肽和抑制剂通过TGF-β调控瘢痕形成。据此，我们在本项目中对ACE相关活性肽及其抑制剂（拮抗剂）进一步作系统的筛选研究，并揭示其调控模式和机制。首先在体外探究了ACE相关活性肽及其抑制剂（拮抗剂）对成纤维细胞活性和功能的影响，并进行药物筛选，这部分实验阐明了ACE为枢纽的活性肽/抑制剂对成纤维细胞的调控作用。再利用筛选得的ACE相关药物制备外用乳膏，在小鼠瘢痕模型上采用局部外用的给药方式进行体内实验，研究ACE相关药物外用对皮肤瘢痕的抑制作用。然后，在此基础上，我们添加壳聚糖和积雪苷，改进辅料配方，并进一步探究ACE外用制剂抑制瘢痕形成的机制。最后，我们采用临床回顾性调查比较了几种市售抗瘢痕外用制剂的效果。结果显示，ACE抑制剂可以减少细胞增殖、胶原沉积和转化生长因子（TGF-β1）的表达，所制备的外用乳膏可以改善瘢痕，且添加壳聚糖和积雪苷之后可以获得更好效果。进一步分析显示，乳膏通过TGF-β/Smad和Wnt/β-catenin两条通路来影响皮肤瘢痕形成。该研究将拓展对瘢痕形成机制和治疗的认识，为下一步制备靶向ACE的抗瘢痕药物提供理论依据。