基于糖基化修饰角度探讨活血化瘀法对特发性肺纤维化的干预机制

To establish multi-target treatment strategies for idiopathic pulmonary fibrosis(IPF),our research group take the proposal which is motivated by previous work from our lab that inhibition of TGF-β receptor subtypes glycosylation could inhibit IPF by suppressing the TGF-β signaling pathway, and according to Traditional Chinese Medicine（TCM） treatment- - Huoxue Huayu Recipe,we have found that TCM- Huoxue Huayu Recipe could inhibit core fucose glycosylation of lung fibroblast cell in vitro and might reduce IPF in vivo，above all these are the basises of the present subject.This project will use advanced technologies such as molecular biology,glycobiology,proteomics etc.,and perform in vivo and in vitro to:(1) elucidate the effect and mechanism (such as oxidative stress, lung fibroblast transdifferentiation,extracellular matrix accumulation,fucose glycosylation, etc.)of inhibiting IPF of Huoxue Huayu Recipe;(2) find out the possibility that Huoxue Huayu Recipe can enhance the efficacy of N-acetylcysteine and pirfenidone,and the possible mechanism;(3) demonstrate target protein and related signal transduction pathway of Huoxue Huayu Recipe on pulmonary fibrosis by the aid of iTraq proteomics and Bioinformatics technology.This subject can make the mechanism of glycosylation on IPF study deeply, and provide the scientific basis for the clinical transformation of integrative medicine of multi-target treatment strategies for IPF.

为建立多靶点联合治疗特发性肺纤维化（IPF）策略，课题组在发现抑制TGF-β受体亚型的糖基化可以抑制TGF-β信号通路从而阻抑IPF的基础上，以中医"活血化瘀"法治疗肺痹证为指导，初步发现活血化瘀方剂具有抑制体外培养的肺成纤维细胞糖基化的作用、减轻动物IPF的可能的前提下提出本课题。本课题中将利用分子生物学、糖生物学、蛋白组学等先进技术，从细胞水平、动物实验两个层次研究（1）活血化瘀方剂对IPF的抑制作用及作用机理（如氧化应激、肺成纤维细胞转分化、细胞基质积聚、糖基化等）；（2）活血化瘀方剂是否可以增强N-乙酰半胱氨酸、吡非尼酮的治疗IPF的疗效及可能机理；（3）利用iTraq蛋白质组及生物信息学技术阐明活血化瘀方剂治疗IPF的可能靶蛋白或关键信号通路。使糖基化修饰在IPF中作用机理得以深入研究，为临床制定中西医结合的多靶点治疗IPF策略提供基础。

肌成纤维细胞是肺间质纤维化进展的重要事件，探索肌成纤维细胞的来源及寻求有效干预策略是延缓肺间质纤维化进展的重要环节。我们的前期工作基础及新近研究表明中药活血化瘀方剂对肺间质纤维化有明显的保护作用，并初步筛选出对肺间质纤维化有保护作用的有效单体成分（丹参酮ⅡA、红景天苷、甘草酸）。在此创新性工作基础上，我们发现EMT是致肺间质纤维化进展的重要途径，丹参酮ⅡA、红景天苷、甘草酸可以有效阻止EMT进程。为进一步明确其机制，我们构建博来霉素诱导的肺间质纤维化大鼠模型，利用蛋白质组学分析筛选出致肺间质纤维化发生的关键蛋白，发现TGF-βR、Wnt等多数蛋白均为糖蛋白，且受到核心岩藻糖基化修饰（core fucosylation, CF）。丹参酮ⅡA、红景天苷、甘草酸可以通过调控CF抑制TGF/smad2/3, wnt/β-catenin的活化，进而干预肺间质纤维化的进展。