SIRT1促进自噬流减轻下肢缺血再灌注损伤的分子机制

Ischemia reperfusion (I/R) injury plays an important role in worsening the prognosis of acute limb ischemia, but the underlying mechanism remains unclear. Our preliminary study found that the protein expression of ATG7, which promotes the formation of autophagosome, and LAMP2, which maintains the function of lysosome, decreased, while the expression of LC3-II, an indicator of autophagosome, inceased, suggesting possible impaired autophagic flux, which was associated with exacerbated limb skeletal muscle injury. Autophagic flux is closely related with energy changes, and further study revealed that SIRT1, a key energy sensor, decreased in line with ATG7 and LAMP2. Thus, we propose that SIRT1 alleviates limb I/R injury by enhancing autophagic flux via coordinated regulation of ATG7 and LAMP2. This project aims to dynamically monitor the number of the autophagosome with lysosome inhibitor and GFP-LC3, in order to prove that impaired autophagic flux is attributable to exacerbated limb I/R injury. Adenoviral expression system and autophagy agonist/inhibitor will then be applied to investigate if SIRT1 reduces the limb I/R injury through increasing autophagic flux. Finally, co-IP, deacetylation site mutations, and adenoviral expression system will be used to explore the potential pathway of how SIRT1 regulates the autophagic flux. This study aims to elucidate the mechanism of enhanced autophagic flux regulated by SIRT1, and to explore novel therapeutic target for mitigating the limb I/R injury.

缺血再灌注（I/R）损伤是急性下肢缺血预后不良的重要原因，但机制未明。我们前期发现：促进自噬体形成的ATG7和维持溶酶体功能的LAMP2表达均下降，而自噬体标志物LC3-II表达增加，提示自噬流可能受损，并与下肢骨骼肌损伤加重相关。自噬流与能量变化密切相关，进一步实验发现能量感受器SIRT1表达下降，并与ATG7和LAMP2变化一致。由此我们推测：SIRT1通过调控ATG7和LAMP2协同促进自噬流，减轻下肢I/R损伤。本项目拟使用溶酶体抑制剂和GFP-LC3监测自噬体数量的动态变化，证实自噬流受损是下肢I/R损伤的重要机制；并通过腺病毒表达技术和自噬通路激动/拮抗剂，研究SIRT1能否促进自噬流减轻I/R损伤；最后采用免疫共沉淀、去乙酰化酶活性域突变和腺病毒表达等技术探索SIRT1调控自噬流的可能通路。本项目旨在阐明SIRT1调控I/R自噬流的机制，为减轻下肢I/R损伤提供新的干预靶点。

缺血再灌注（I/R）损伤是急性下肢缺血预后不良的重要原因，但机制未明。根据我们前期的发现，我们推测：SIRT1通过调控ATG7和LAMP2协同促进自噬流，减轻下肢I/R损伤。本项目旨在阐明SIRT1调控I/R自噬流的机制，为减轻下肢I/R损伤提供新的干预靶点。.由于关键设备障碍导致细胞水平建模滞后，本项目部分体外试验未能如期进行，将会在后续研究中继续进行。现已完成主要研究内容有：1)证实了自噬流激活差异对下肢I/R损伤有明显影响；2)证实了增强自噬流可以对下肢I/R损伤起到有效的保护作用。.重要结果、关键数据：1)成功将C2C12细胞系（肌原细胞）诱导分化出成熟的骨骼肌细胞；2)成功构建了小鼠下肢单纯缺血和下肢I/R模型。通过Triphenyl Tetrazolium Chloride Assay，TUNEL，扫描电镜与west blotting，我们发现下肢I/R损伤组相比于下肢单纯缺血自噬体较少，但相对其损伤更为严重。3)通过使用自噬增强剂雷帕霉素与自噬抑制剂3-MA，进而观察增强自噬流与抑制自噬流对下肢I/R损伤的作用。通过扫描电镜，west blotting，Triphenyl Tetrazolium Chloride Assay与TUNEL，证实了使用雷帕霉素这一自噬增强剂可以有效增强自噬流并对小鼠下肢I/R损伤起到有效保护作用。.科学意义：I/R损伤是下肢ALI预后不良的重要原因，但目前机制尚未清楚，并且缺乏切实有效的治疗手段。本项目通过研究自噬流在下肢I/R损伤，证实了自噬流激活差异对下肢I/R损伤有明显影响，并进一步证实了调控自噬流可以对下肢I/R损伤有明显的影响。我们从自噬的细胞生物学机制角度研究了下肢I/R损伤的机制，希望发掘有效减轻下肢I/R损伤的新方法，从而改善我国众多ALI患者的预后，具有重要的临床价值和社会效益。