The Protective Effect And Molecular Mechanism Of Thioredoxin-1 On Ischemia-reperfusion Skin Flap

Objectives:Ischemia-reperfusion injury is inevitable during free-flap transfer, causing postoperative circulatory disorder, tissue edema, and flap infarction. Currently, skin flap ischemia-reperfusion injury is widely acknowledged to be closely related to extensive oxidative stress and cell apoptosis induced by the reactive oxidative species burst in the flap tissue. Thioredoxin (Trx) is a fundamental protein which is extensively expressed in a variety of human tissues and cells, and is essential for maintaining the balance of the cellular redox status and regulating the apoptosis signaling pathways. Our previous study revealed the gene trxl is correlated to the skin tissue resistance against hypoxia. Therefore, the main purpose of this research is to investigate the protective effects and molecular mechanisms of Trx-1 on ischemia-reperfusion skin flaps and its prognostic and therapeutic value by clinical sample test, in vitro and in vivo study.Methodology:1. Ten clinical specimens of ischemia-reperfusion flaps and normal controls were collected from the patients undergoing free flap transfer. The human samples were assessed for apoptosis and Trx-1 expression by TUNEL assay and immunohistochemistry. The linear regression analysis was used to study the correlation between Trx-1 expression and apoptosis.2. An in vitro hypoxia model of the keratinocyte (HaCAT cell lines) was induced by CoCl2. The apoptosis-related protein expression of HaCAT cells under the condition of hypoxia was tested. The protective effects and molecular mechanisms of recombinant human Trx-1 on the hypoxic keratinocytes were determined.3. A mouse ischemia-reperfusion flap model was established. The flap survival rate difference was observed and recorded within 7 days after reperfusion. The flap tissues were harvested and tested by hematoxylin-eosin and immunohistochemistry staining, TUNEL assay, SOD and MDA measurement, and Western blot. The protective effects and molecular mechanisms of Trx-1 on ischemia-reperfusion skin flaps and its prognostic and therapeutic value were determined.Principal findings:1. The apoptosis index increased and the Trx-1 expression decreased significantly (p< 0.01) in human ischemia-reperfusion skin flap samples. The Trx-1 abundance was negatively correlated with IR-induced apoptosis in human samples (r2= 0.89, p< 0.01).2. Trx-1 depletion in HaCAT cells was induced by hypoxia (p< 0.01), while the expression of ASK1, p-p38, and the apoptosis marker c-PARP was significantly upregulated (p< 0.01).3. The recombinant human Trx-1 pretreatment significantly enhanced the Trx-1 expression in HaCAT cells (p< 0.01) and reduced the abundance of apoptosis-related proteins HIF-la, p-p38, and c-PARP (p< 0.05).4. The apoptosis index increased and the Trx-1 expression decreased significantly (p< 0.01) in mice ischemia-reperfusion skin flaps compared with sham controls. The Trx-1 abundance was negatively correlated with IR-induced apoptosis in mice samples (r2= 0.93,p< 0.01).5. The pathological damage of the ischemia-reperfusion flaps in mice was mitigated significantly (p< 0.01) and the survival rate increased significantly (p< 0.01) following recombinant human Trx-1 pretreatment.6. A significant increase of SOD activity and decrease of MDA concentration were observed in mice ischemia-reperfusion flaps compared with sham controls (p< 0.01). The recombinant human Trx-1 pretreatment significantly (p< 0.05) enhanced the SOD activity and reduced the MDA concentration of the ischemia-reperfusion flaps.7. Trx-1 depletion and a significant increase in ASK1, p-p38, and cl-caspase-3 abundance was revealed in mice ischemia-reperfusion flaps (p< 0.01), while supplementation of the recombinant human Trx-1 significantly upregulated the Trx-1 abundance (p< 0.01) and reduced the expression of apoptosis-related proteins ASK1, p-p38, and cl-caspase-3 (p< 0.05).Conclusions:1. Trx-1 plays a significantly protective role in skin flap ischemia-reperfusion injury and keratinocyte hypoxia.2. Trx-1 exerts the protective function through its antioxidant and antiapoptotic activities of directly scavenging the reactive oxidative species and inhibiting the ASK-MAPK signaling pathway.3. Trx-1 may be an imperative prognostic marker of ischemia-reperfusion flap viability after free flap transfer.4. Trx-1 may serve as a potential therapeutic target for skin flap ischemia-reperfusion injury in clinical application.