间隙连接蛋白43模拟肽aCT1对移植肝缺血再灌注损伤的保护作用及机制研究

Ischemia reperfusion injury (IRI) is inevitable in clinical liver transplantation. Reducing IRI are key factors in improving the prognosis of grafts. Cellular gap junction and tight junction play an important role in maintaining normal cell-cell communication and barrier function, so it is possible to protect cell junctional integrity for reducing IRI during transplantation. Connexin43 (Cx43) mimetic peptide alpha connexin43 carboxy-terminus peptide 1 (aCT1) mediated inhibition of Cx43 binding with zonula occludens-1 and had been shown to reduce inflammation and promote chronic skin ulcer wound healing in clinic trial. Previous studies have confirmed that aCT1 can stabilize the gap junction and tight junction on endothelial cells, reduce IRI, and have a protective effect on heart/lung transplant IRI in mice. Then we found that aCT1 could reduce the cold preservation damage in liver graft. Therefore, we hypothesize that aCT1 stabilizes the gap junction and tight junction in hepatic sinusoidal endothelial cells and hepatocytes, and reduces inflammatory cytokines release and inflammatory cells infiltration. The aim of our study is to investigate the feasibility and mechanism of aCT1 ameliorates liver transplant IRI in vivo, cellular and molecular levels by orthotopic liver transplantation model with 24h cold storage in mice, which provides a new strategy for prevention and treatment of liver IRI in clinical transplantation.

缺血再灌注损伤（IRI）在临床肝移植过程中无法避免，减轻IRI是改善移植物预后的重要因素之一。由于细胞连接在维持细胞间通讯和屏障功能中起重要作用，通过干预细胞连接防治肝移植IRI成为可能。间隙连接蛋白43模拟肽aCT1，可抑制间隙连接蛋白43与紧密连接闭锁蛋白-1的结合，临床试验表明可减少炎症反应，促进皮肤溃疡创面的愈合。申请者前期研究证实，aCT1可通过稳定内皮细胞的间隙连接与紧密连接而减轻细胞IRI，亦对小鼠心脏及肺移植物IRI具有保护作用，近期预实验发现aCT1可减轻肝脏的冷保存损伤。据此推测，aCT1可能通过稳定肝窦内皮细胞和肝细胞的间隙连接与紧密连接，减少炎性细胞因子释放及炎性细胞浸润，从而减轻移植肝IRI。本项目拟借助供肝经历24小时冷保存的小鼠同系原位肝移植模型，分别从整体、细胞及分子水平，探究aCT1对移植肝IRI的保护作用及相关机制，为临床防治移植肝IRI提供新的策略。

缺血再灌注损伤（IRI）在临床肝移植过程中无法避免，减轻IRI是改善移植物预后的重要因素之一。由于细胞连接在维持细胞间通讯和屏障功能中起重要作用，通过干预细胞连接防治肝移植IRI成为可能。前期研究发现间隙连接蛋白43模拟肽aCT1可通过稳定内皮细胞的间隙连接与紧密连接而减轻细胞IRI。本项目通过供肝经历24小时冷保存的小鼠同系原位肝移植模型，发现可明显提高供肝的保存质量，减轻移植肝的缺血再灌注损伤。act1组术后6h血清ALT、AST及LDH较UW对照组明显降低，分别为(2021±655 vs 4585±1222)U/L、(1996±650 vs 5518±1235)U/L、(6597±1785 vs 29001±7112)U/L；病理HE染色提示act1组移植肝肝窦结构完好，肝细胞未见明显变性坏死；透射电镜结果提示act1组肝细胞间隙连接结构完整。其机制可能是通过上调肝组织Cx43蛋白的表达，并促进其磷酸化；减少缺血再灌注损伤对肝细胞间紧密连接完整性的破坏，稳定紧密连接蛋白Occludin及ZO-1的表达，维持细胞连接完整性，并减少内皮细胞的损伤及肝内炎性细胞的浸润与活化。本研究对act1在临床器官保存的方面的应用提供了动物实验数据，为进一步提高临床移植肝保存水平及供体质量提供新的策略。