MG53/Caveolin-1细胞膜修复复合体调控肝细胞Ferroptosis在肝缺血再灌注损伤中的作用及机制研究

Hepatic ischemia-reperfusion injury (HIRI) often leads to postoperative liver dysfunction and directly affects the prognosis of patients，which has no effective prevention and treatment. Oxidative stress injury plays a dominant role in HIRI. Applicant have found that HIRI-induced oxidative stress injury and reactive oxygen species (ROS) accumulation in hepatocyte. Although the protective effects of antioxidants are not obvious, MG53 has obvious protective effects on repairing cytomembrane. In pre-experiment, we further found that ROS accumulation caused hepatocyte Ferroptosis, MG53 and CAV-1 formed a cytomembrane-repair complex to reduce Ferroptosis, which suggesting that repairing cytomembrane damage and blocking Ferroptosis caused by ROS accumulation become the main point of HIRI prevention and treatment. According to this project, we hypothesize that "MG53/CAV-1 cytomembrane-repair complex alleviate HIRI by regulating hepatocyte Ferroptosis". The mouse HIRI model and hepatocyte hypoxia-reoxygenation model were constructed by TAT-MG53 fusion protein technology and in vivo knockdown of mouse MG53 protein technology. The mechanism of prevention and treatment of HIRI will be studied from the perspective of membrane repair. It will provide a new theoretical basis and targets.

肝缺血再灌注损伤（HIRI）常导致术后肝功能不全并直接影响患者预后，目前尚无有效防治手段。氧化应激损伤是HIRI的关键机制，申请人既往研究发现HIRI致肝细胞氧化应激损伤，活性氧（ROS）在细胞内蓄积，应用抗氧化剂效果不明显，MG53修复细胞膜却有明显的保护作用。进一步预实验发现ROS堆积致肝细胞发生Ferroptosis，补充MG53重组蛋白可减轻Ferroptosis，同时检测到此过程MG53与陷窝蛋白Caveolin-1结合紧密，抑制Caveolin-1削弱了其保护作用。据此本项目提出“MG53/Caveolin-1细胞膜修复复合体通过调控肝细胞Ferroptosis减轻HIRI损伤”的假说。拟通过TAT-MG53融合蛋白、在体敲减小鼠MG53蛋白等技术，构建小鼠HIRI模型及肝细胞缺氧复氧模型，从膜修复角度探讨防治HIRI的机制，为遏制HIRI后肝细胞损伤提供理论依据及新的靶点。

肝缺血再灌注损伤（HIRI）常导致术后肝功能不全并直接影响患者预后，氧化应激损伤造成的活性氧（ROS）大量堆积是HIRI早期的关键机制，但目前尚无有效的干预措施。本课题在构建C57BL/6小鼠HIRI模式的基础上，首先探讨再灌注早期不同时间点内肝细胞损伤与氧化应激产物及Ferroptosis之间的关系，同时检测了细胞膜修复蛋白MG53在肝脏的表达情况，选取再灌注后6h作为后续实验的时间节点。我们成功构建C57BL/6小鼠肝脏MG53在体敲除模型，通过尾静脉高动力大剂量注射MG53 shRNA重组腺病毒载体的方法，实现了再体干预肝脏MG53蛋白表达的可能，并且发现该方法对其他器官（如骨骼肌、心肌等）影响较小，实现了在体干预小鼠肝细胞MG53蛋白表达的可能。同时，我们合成TAT-MT53融合蛋白，通过细胞实验和在体检测等，验证了TAT-MG53融合蛋白在C57BL/6小鼠体内的细胞毒性、有效剂量及作用时间。证实尾静脉注射6.0mg/kg剂量的融合蛋白在小鼠肝脏表达的峰值出现在注射后8h，并将这一结果将作为后续研究的参考。由此，我们通过干预C57BL/6小鼠肝细胞内MG53蛋白的表达，证实HIRI过程中，MG53蛋白通过抗氧化应激的作用，减轻了肝细胞的Ferroptosis，起到肝脏保护的作用。另外，我们发现使用Caveolin-1抑制剂Daidzein后，MG53保护HIRI的作用被取消，证实在缺血再灌注的肝脏，MG53蛋白通过上调CAV-1的表达发挥作用。我们研究从新的角度为遏制HIRI后肝细胞损伤提供理论依据及新的靶点。