TFEB介导的自噬流修复在皮瓣缺血再灌注损伤中的作用及调控机制研究

Necrosis of the transplanted skin flap is mainly due to ischemia-reperfusion (I/R) injury. However, there is a lack of effective treatment. Previously we demonstrated that autophagy flux was impaired at the early stage of I/R injury in skin flap. To further understand the mechanism of this phenomenon, we will verify whether reactive oxygen species (ROS) can contribute to lysosome functional impairment and further the blockade of autophagy flux. It was reported that the impairment of autophagy flux can result in cell damage. It was found that transcriptional factor TFEB regulation can reduce the level of ROS and promote the activities of lysosome and autophagosome, consequently, repair autophagy flux. Besides, our preliminary data showed that the activity of TFEB was significantly enhanced in I/R injury of skin flap. Therefore, we will regulate TFEB activity to promote the repair of autophagy flux, and further alleviate I/R injury of skin flap. Furthermore, we will explore whether the activity of TFEB can be regulated by the PARP1-LKB1-AMPK-mTOR signaling pathway. Finally, we will demonstrate the protection of TFEB induced autophagy and the PARP1-LKB1-AMPK-mTOR signaling pathway for TFEB activity regulation. This study is to elucidate a new mechanism of autophagy flux blockade in I/R injury of skin flap， and to provide a strategy of taking the regulation of TFEB activity to repair autophagy flux as a target to alleviate I/R injury of skin flap.

缺血再灌注(I/R)损伤是移植皮瓣坏死的主要原因，但缺乏有效的治疗手段。前期发现， I/R损伤皮瓣早期发生自噬流阻滞。为明确该现象的病理机制，本研究将在皮瓣I/R损伤中，验证活性氧簇（ROS）通过破坏溶酶体功能，导致自噬流的阻滞。而自噬流的障碍能造成细胞损害。转录因子TFEB的调控能通过降低ROS水平、增强溶酶体及自噬体活性，促进自噬流修复。前期发现，TFEB的活性在皮瓣I/R损伤中显著增强。本研究将探究通过调控TFEB活性促进自噬流修复，以治疗皮瓣I/R损伤。再考证I/R损伤皮瓣中TFEB受PARP1-LKB1-AMPK-mTOR信号通路的调控。最后，在体外人体细胞模型中，证明PARP1-LKB1-AMPK-mTOR通路对TFEB的调控及TFEB介导的自噬激活对细胞的保护作用。本项目旨在为皮瓣I/R损伤阐明自噬流阻滞的新机制；并提出通过TFEB调控修复自噬流，治疗皮瓣I/R损伤的新策略。

缺血再灌注损伤是皮瓣移植术后坏死的主要原因和机制，且防治移植皮瓣缺血再灌注损伤真正可行的治疗方案并不多。探究如何有效减轻或避免缺血再灌注损伤成为目前皮瓣外科探索的主要方向。但在皮瓣缺血再灌注损伤中，自噬所扮演的角色尚不得而知。本项目首先验证在皮瓣缺血再灌注损伤中，ROS破坏细胞溶酶体功能，进而导致自噬流的阻滞。然后，探究通过调控细胞内 TFEB 活性，促进自噬流修复，以治疗皮瓣的缺血再灌注损伤。再者，进一步考证缺血再灌注皮瓣中，TFEB 活性受 PARP1-LKB1-AMPK-mTOR 信号通路的调控。最后，在体外人体细胞模型中，证明 PARP1-LKB1-AMPK-mTOR 信号通路对 TFEB 活性调控及 TFEB 介导的自噬激活对细胞的保护作用。研究发现缺血再灌注损伤皮瓣中ROS诱发细胞溶酶体功能失代偿及自噬流阻滞。其次，通过调控转录因子 TFEB 活性，修复自噬流阻滞，从而治疗皮瓣缺血再灌注损伤。此外，阐明皮瓣缺血再灌注损伤中信号通路对TFEB 活性的调控机制。在动物及细胞实验中，验证缺血再灌注损伤皮瓣细胞内 TFEB 活性受PARP1-LKB1-AMPK-mTOR 信号通路的调控。 最后，研究发现多种抑制氧化应激，激活TFEB的药物对缺血皮瓣有显著促存活作用。具体如下，二甲双胍、海藻糖分别促进随意皮瓣远端区域的存活，并伴随组织内细胞自噬的激活，且通过减少氧化应激，激活TFEB通路发挥作用；丹参酚酸B通过激活细胞自噬，以促进超长随意皮瓣的存活；成纤维细胞生长因子21（FGF21）激活TFEB介导细胞自噬，以促进超长随意皮瓣的存活，且FGF21可能通过激活AMPK-mTOR通路发挥作用等。本项目首次成果阐明 ROS 诱导皮瓣缺血再灌注损伤中的溶酶体活性抑制及自噬流阻滞的新机制。提出通过调控转录因子 TFEB 活性，修复自噬流阻滞，以治疗皮瓣缺血再灌注损伤的新理论。通过调控TFEB介导的自噬流，为皮瓣缺血再灌注损伤治疗提供一种新的思路。该成果对于将来皮瓣缺血再灌注损伤防治的临床方法提供新策略。