HIF相关正反馈回路及其信号通路在瘢痕疙瘩发生与发展中的作用及分子机制

Keloid is a common type of pathological skin healings, but its pathogenesis remains poorly understood. The recent literatures reported that hypoxia and hypoxia inducible factor (HIF) activation during healing of corium-involved tresis vulnus are frequently presented, and HIF up-regulates the expression of connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-β) and inducible nitricoxide synthase (iNOS). Our preliminary study found that the HIF-1, Toll-like receptor 4 (TLR4) and nitrogen monoxidum levels in keloid tissue were significantly increased, but the correlation and possible active mechanism of these findings referring to keloid generation are unknown. In this project, we presumed that HIF-1 and HIF-2 mediate anoxic stress reaction to cause the generation and development of keloid. We will try to elucidate the molecular mechanism of keloid formation from different levels of clinical specimen, molecule and cell, and animal model. We will determine that the HIF-1 or HIF-2 and TGF-β positive feedback loop to activate Smads pathway and up-regulate CTGF expression, and the HIF-1 or HIF-2 and NO or TLR4 positive feedback loop to activate NF-κB, p38MAPK and ERK1/2 pathways, which cause keloid formation through high expression of collagen proteins and mucoitin, and oversynthesis and deposition of ECM. Moreover, we will also determine the role and diversity of HIF-1 and HIF-2 during keloid formation with different anoxic degrees. The findings of this project will possess high originality and medical significance.

瘢痕疙瘩是常见病理性皮肤愈合类型，但发病机制至今未完全明了。近年文献报道累及真皮的创口愈合时常呈缺氧状态及缺氧诱导因子（HIF）激活，上调结缔组织生长因子（CTGF）、TGF-β和诱导型NO合酶表达。我们前期研究发现瘢痕疙瘩HIF-1和HIF-2、TLR4表达及NO水平显著升高，但与瘢痕疙瘩关系及作用机制不明。本项目中我们将从HIF介导缺氧应激反应而导致瘢痕疙瘩发生与发展的思路出发，从临床标本、分子与细胞、动物模型不同层次确定：HIF-1或HIF-2与TGF-β形成正反馈回路激活Smads通路或上调CTGF表达，HIF-1或HIF-2与NO或TLR4形成另一正反馈回路并激活NF-κB、p38MAPK和ERK1/2通路，引起胶原蛋白高表达、粘多糖和ECM过度合成与沉积而引发瘢痕疙瘩分子机制；同时确定不同缺氧程度时HIF-1和HIF-2在瘢痕疙瘩形成中的作用及其差异，具有较高创新性和临床意义。

项目背景：瘢痕疙瘩是常见病理性皮肤愈合类型，但发病机制至今未完全明了。近年文献报道累及真皮的创口愈合时常呈缺氧状态及缺氧诱导因子（HIF）激活，上调结缔组织生长因子（CTGF）、TGF-β和诱导型NO合酶表达。我们前期研究发现瘢痕疙瘩 HIF-1 和 HIF-2、TLR4表达及NO水平显著升高，但与瘢痕疙瘩关系及作用机制不明。.主要研究内容：从临床标本、分子与细胞、动物模型不同层次研究：HIF对TGF-β/Smads通路和NF-κB、p38MAPK和ERK1/2通路的作用，进而引起胶原蛋白过度合成与沉积而引发瘢痕疙瘩的分子机制。.重要结果：.1. 免疫组化显示瘢痕疙瘩组织中 HIF-1α 的表达明显高于正常皮肤组织。.2. 在正常皮肤和瘢痕疙瘩成纤维细胞中，缺氧激活了TGF-β/Smad信号通路。1% O2 缺氧24 h，TGF-β1、smad2/3、p-Smad2/3、Smad4在mRNA和蛋白水平均升高，且p-Smad2/Smad2、p-Smad3/Smad3升高。而在缺氧条件下抑制HIF-1α则能使TGF-β1、smad2/3、p-Smad2/3、Smad4均降低且p-Smad2/Smad2、p-Smad3/Smad3降低。.3. HIF-1α和TGF-β1/Smad通路共同促进了胶原沉积。缺氧促进胶原沉积，而在缺氧条件下抑制HIF-1α的表达能减少胶原沉积。同时，在缺氧或常氧条件下抑制TGFβR2均能抑制胶原沉积。.4. HIF-1α 激活 TLR-MyD88-NF-κB 通路及下游 IL-6 的表达，促进 ERK1/2 磷酸化及下游 IL-8 的表达，促进 TLR-MyD88-NF-κB 通路与 TGF-β/Smad 通路的crosstalk 作用。.5. HIF-1α 抑制剂（ 2-Methoxyestradiol (2-ME2)）在裸鼠体内对瘢痕疙瘩具有一定的抑制作用。.结论：在皮肤成纤维细胞中缺氧诱导的HIF-1α激活了TGF-β1/Smad和TLR-MyD88-NF-κB 、ERK1/2信号通路； HIF-1α与TGF-β1/Smad信号通路共同促进了细胞胶原的沉积；HIF-1α诱导的TLR/MyD88/NF-κB通路促进了TGF-β/Smad通路的激活。结合动物实验，证明了HIF-1α对瘢痕疙瘩的发生和发展具有促进作用。