第一部分：丙泊酚通过调控铁死亡抑制乳腺癌细胞增殖并增强其对化疗药的敏感性

背景：乳腺癌是女性最常见的恶性肿瘤之一，而三阴性乳腺癌（Triple-negative breast cancer, TNBC）则是其中发病率高、恶性程度高、预后差的临床类型之一。手术切除仍然是乳腺癌的首选治疗方案，作为临床上围术期最常用的静脉麻醉药之一，丙泊酚对乳腺癌的影响及其潜在机制目前尚存在争议，而且研究大多局限在细胞凋亡领域；此外，只有少数研究探讨了丙泊酚对乳腺癌细胞化疗敏感性的影响，更缺少针对TNBC的研究。铁死亡是近年来新发现的一种细胞程序性死亡方式，已被证实参与体内多种病理生理状态。因此，本研究旨在探索丙泊酚及其常用临床制剂是否通过调节细胞铁死亡来影响TNBC细胞的增殖及其对化疗药的敏感性。

方法：本研究采用TNBC细胞系MDA-MB-231，选择丙泊酚原料药、丙泊酚注射乳剂（Propofol injectable emulsion, PIE）以及磷丙泊酚钠三种丙泊酚制剂，将其单独或联合化疗药（多柔比星，紫杉醇）应用，处理细胞24 h后，采用CCK-8试剂盒检测细胞活力，采用流式细胞技术、免疫印迹试验（Western blot）、多重荧光免疫组化（Multiplex immunohistochemistry, mIHC）、透射电子显微镜来评估细胞的凋亡水平，采用相应试剂盒检测细胞内活性氧（Reactive oxygen species, ROS）和Fe²⁺水平，采用透射电子显微镜观察细胞铁死亡特征性线粒体形态学变化，采用Western blot和mIHC检测铁死亡相关调节蛋白（p53，SLC7A11，GPX4，FSP1，Ubiquinone，Ubiquinol）的表达水平。

结果：CCK-8细胞活力检测结果表明丙泊酚原料药可以显著抑制MDA-MB-231细胞的增殖，而PIE和磷丙泊酚钠的作用不明显；三种丙泊酚制剂均增强了多柔比星和紫杉醇对MDA-MB-231细胞的抑制作用。细胞凋亡的检测结果提示丙泊酚的确可以促进MDA-MB-231细胞的凋亡，但其对细胞增殖的抑制作用并不能完全由凋亡来解释。随后对于细胞内ROS水平、Fe²⁺水平的测定，以及线粒体形态学的观察和铁死亡相关调节蛋白的测定显示，无论是单独应用还是联合应用化疗药，三种不同制剂的丙泊酚可以诱发铁死亡相关改变，包括诱导线粒体发生相应的形态学改变，促进细胞内ROS和Fe²⁺蓄积，并且这种调节可能是通过p53-SLC7A11-GPX4通路实现的。

结论：丙泊酚可以通过促进细胞铁死亡从而抑制TNBC细胞的增殖并增强其对化疗药的敏感性，这种调节作用可能是通过p53-SLC7A11-GPX4通路实现的。

第二部分：丙泊酚注射乳剂通过调控铁死亡参与改善肝细胞的缺血/再灌注损伤

背景：肝脏缺血/再灌注（Ischemia/reperfusion, I/R）损伤是肝脏手术后常见的并发症，可导致预后不良、住院时间延长和住院费用增加。近年来的研究发现，铁死亡参与并促成肝脏I/R损伤，靶向抑制铁死亡可能成为改善肝脏I/R损伤的新策略。丙泊酚注射乳剂（Propofol injectable emulsion, PIE）是围术期最常用的静脉麻醉药之一，根据既往文献报道和我们前期的研究结果，我们推测PIE中的外源性脂质成分可能存在铁死亡抵抗和保护作用。因此，本研究以肝细胞I/R损伤模型为载体，探究PIE是否可以通过抑制肝细胞铁死亡而缓解其I/R损伤，希望为改善围术期肝脏I/R损伤、减少术后并发症提供新的靶点，为围术期器官保护提出新的管理策略。

方法：本研究采用正常大鼠肝细胞系IAR20进行实验，并对其进行缺氧/复氧（Hypoxia/reoxygenation, H/R）建模，模拟I/R损伤；另一方面我们将PIE拆分成丙泊酚原料药和脂肪乳剂，同时比较丙泊酚原料药、PIE、脂肪乳剂注射液在IAR20细胞I/R损伤中的影响，从“PIE对肝细胞I/R损伤的作用”、“PIE对肝细胞铁死亡的影响”、以及“PIE对肝细胞I/R损伤的作用是否通过铁死亡实现”这三部分进行探索。我们采用CCK-8试剂盒检测细胞活力，采用透射电子显微镜观察细胞线粒体形态，采用线粒体膜电位检测试剂盒JC-1分析线粒体膜电位和功能，采用DCFDA/H2DCFDA和FerroOrange试剂盒分别测定细胞内活性氧 (Reactive oxygen species, ROS) 和Fe²⁺水平。

结果：我们的结果表明，首先，与丙泊酚原料药相比，PIE和脂肪乳剂注射液可以改善H/R损伤对IAR20细胞活力的抑制。其次，PIE和脂肪乳剂可以改善铁死亡诱导剂Erastin引起的IAR20细胞活力下降、铁死亡特征性线粒体形态改变、线粒体膜电位受损、以及细胞内ROS和Fe²⁺的堆积，即缓解Erastin诱导的IAR20细胞铁死亡，而丙泊酚原料药则未表现出此作用。第三，PIE和脂肪乳剂可以改善H/R损伤后导致的IAR20细胞活力下降、铁死亡特征性线粒体形态改变、线粒体膜电位受损、ROS和Fe²⁺堆积，且与铁死亡抑制剂Ferrostatin-1作用一致，而丙泊酚原料药却没有表现出类似的作用。

结论：PIE可以通过抑制细胞铁死亡对肝细胞I/R损伤起到保护作用，这种保护作用主要来自于PIE中的脂肪乳剂成分，而非丙泊酚药物成分，PIE中外源性的脂肪乳剂成分确实存在铁死亡抵抗作用。

Part I: Propofol inhibits proliferation and augments the anti-tumor effect of doxorubicin and paclitaxel partly through promoting ferroptosis in triple-negative breast cancer cells

Background: Triple-negative breast cancer (TNBC) is relatively common in women and is associated with a poor prognosis after surgery and adjuvant chemotherapy. Currently, the mechanism underlying the relationship between propofol and breast cancer is controversial and limited to cell apoptosis. Moreover, there are only a few studies on the effect of propofol on the chemotherapeutic sensitivity of TNBC cells. Therefore, this study explored whether propofol and its commonly used clinical formulations affect the proliferation and chemotherapeutic effects on TNBC cells by regulating cell ferroptosis.

Methods: We selected MDA-MB-231 cells, which is a kind of TNBC cell lines, treated with propofol, propofol injectable emulsion (PIE) and fospropofol disodium, alone or combined with doxorubicin or paclitaxel to conduct this study. The cell viability, apoptosis, intracellular reactive oxygen species (ROS) accumulation, ferroptosis-related morphological changes, intracellular Fe²⁺ levels, and the expression and localization of ferroptosis-related proteins (p53, SLC7A11 GPX4, FSP1, Ubiquinone, Ubiquinol) were investigated using CCK-8 kit, flow cytometry, DCFDA/H2DCFDA-cellular ROS assay kit, transmission electron microscopy, FerroOrange iron assay, western blot, and multiplex immunohistochemistry (mIHC). 

Results: We found that propofol significantly inhibited MDA-MB-231 cell proliferation, and all three propofol formulations augmented the anti-tumor effects of doxorubicin and paclitaxel. The results from the ROS assay, transmission electron microscopy, intracellular Fe²⁺ assay, western blot, and mIHC revealed that propofol not only induced apoptosis but also triggered ferroptosis-related changes, including morphological changes of mitochondria, increased intracellular ROS levels, and intracellular iron accumulation in MDA-MB-231 cells. The ferroptosis-related p53-SLC7A11-GPX4 pathway was also altered under different treatment propofol, doxorubicin, or paclitaxel regimens.

Conclusion: Propofol showed anti-proliferation effects on TNBC cells and could be a potential adjuvant to enhance the chemotherapeutic sensitivity of TNBC cells partly by promoting cell ferroptosis.

Part II: Targeting ferroptosis by propofol injection emulsion protects hepatocytes against ischemia/reperfusion injury

Background: Ischemia/reperfusion (I/R) injury is a common complication after hepatic surgery, which may associated with poor prognosis, prolonged length of stay and increased cost. It is reported that ferroptosis is related with hepatic I/R injury. Inhibition of ferroptosis may become a new strategy to protect patients against hepatic I/R injury. Propofol injectable emulsion (PIE) is one of the most commonly used intravenous anesthetics in clinical practice. According to the previous research and our own results, we speculated that exogenous emulsion in PIE may have protective effect through ferroptosis resistance. In this study, we used hepatocyte I/R injury model to explore whether PIE could alleviate I/R injury by inhibiting ferroptosis of hepatocytes, hoping to provide a new strategy for perioperative organ protection. 

Methods: Normal rat hepatocyte line IAR20 and hypoxia/reoxygenation (H/R) model were used to simulate hepatic I/R injury, and the effects of PIE, propofol, and emulsion on the IAR20 cell line after I/R injury (H/R injury) were compared. Our study mainly had three parts: 1. the effects of PIE on IAR20 cell line after I/R injury; 2. the effects of PIE on the ferroptosis of IAR20 cell line; 3. whether the effects of PIE on IAR20 cell line after I/R injury associated with ferroptosis. The cell viability, ferroptosis-related morphological changes, mitochondrial membrane potential, intracellular reactive oxygen species (ROS) accumulation and intracellular Fe²⁺ levels were investigated using CCK-8 kit, transmission electron microscopy, JC-1 kit, DCFDA/H2DCFDA-cellular ROS assay kit and FerroOrange iron assay kit. 

Results: Our results suggest that, first, PIE and emulsion could improve the IAR20 cell viability after H/R injury, compared with propofol. Second, the analyses from the cell viability, ferroptosis-related morphological changes, mitochondrial membrane potential, intracellular ROS and Fe²⁺ assay revealed that PIE and emulsion could alleviate the ferroptosis of IAR20 cell line induced by Erastin, a famous activator of ferroptosis, but propofol did not show these effects. Third, also through the results of CCK-8 kit, transmission electron microscopy, JC-1 kit, DCFDA/H2DCFDA-cellular ROS assay kit and FerroOrange iron assay kit, we found that PIE and emulsion protected IAR20 cell line against H/R injury, consistent with the inhibitor of ferroptosis, Ferrostatin-1, while propofol did not.

Conclusion: PIE could protect hepatocytes against I/R injury through inhibiting ferroptosis. This protective effect was mainly attributed to the exogenous emulsion in PIE, but not to its propofol component.