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| 铁死亡抑制剂通过保护膜铁转运蛋白减轻脓毒症相关急性肾损伤 |
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张楷齐1, 吴晶魁2, 姜娜1, 林其圣1, 祝旭颖1, 李佳琳1, 邵兴华1, 杨渊婷1, 倪兆慧1
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1.上海交通大学医学院附属仁济医院肾脏科, 分子细胞(肾病)实验室, 上海交通大学医学院尿毒症诊治中心, 上海市腹膜透析研究中心, 上海 200127;2.上海中医药大学附属曙光医院肾病科, 上海 201200
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| 摘要: |
| 目的 利用脂多糖（lipopolysaccharide, LPS）构建脓毒症体内外模型，探讨铁死亡抑制剂在脓毒症相关急性肾损伤（sepsis associated acute kidney injury, S-AKI）中的保护作用及可能机制。方法 构建脓毒症小鼠模型，随机分为 4组：对照组、LPS组、LPS＋铁死亡抑制剂1（Ferrostatin-1, Fer-1）组和LPS＋铁死亡抑制剂2（Deferoxamine, DFO）组，每组3只小鼠。LPS组小鼠腹腔注射LPS（15 mg/kg），铁死亡抑制剂组分别在LPS注射前30 min腹腔注射Fer-1（5 mg/kg）和DFO（100 mg/kg）。24 h后留取小鼠血液和肾脏标本进行肾功能和铁死亡相关标志物检测。体外培养人肾小管上皮细胞（HK-2），检测100 μg/mL LPS刺激后细胞活性和不稳定铁池、脂质过氧化变化情况。使用siRNA转染HK-2细胞敲低膜铁转运蛋白（ferroportin, FPN）后检测铁死亡标志物变化情况。结果 与对照组相比，LPS组小鼠血肌酐、尿素氮明显升高，肾脏病理损伤加重，铁死亡标志物显著增加，而铁死亡抑制剂组可明显缓解LPS诱导的肾脏功能和病理损伤，减少铁死亡表征。同时，Western印迹法和免疫组化结果显示，LPS组体内外模型中FPN表达量均显著下调，使用siRNA沉默HK-2细胞FPN观察到铁死亡表征增加，说明FPN参与铁死亡损伤过程。qRT-PCR结果显示，FPN下调与肾脏铁调素水平升高相关。进一步检测发现LPS激活铁调素启动子IL-6/STAT3通路，而Fer-1与DFO可以抑制相关通路的激活，减少铁调素mRNA表达，避免FPN下调。结论 LPS可诱导FPN下调介导的铁死亡损伤，铁死亡抑制剂可能通过抑制铁调素降解FPN在LPS诱导的S-AKI中发挥保护作用。 |
| 关键词: 铁死亡 脓毒症 急性肾损伤 膜铁转运蛋白 铁调素 |
| DOI：10.12025/j.issn.1008-6358.2023.20230266 |
| 分类号:R459.7 |
| 基金项目:国家自然科学基金面上项目(82070693,81770666),上海市科学技术委员会启明星项目(扬帆专项,20YF1424900,22YF1423400),上海申康医院发展中心“促进市级医院临床技能与临床创新能力三年行动计划”(SHDC2020CR3029B). |
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| Ferroptosis inhibitors attenuate sepsis associated acute kidney injury by protecting ferroportin |
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ZHANG Kai-qi1, WU Jing-kui2, JIANG Na1, LIN Qi-sheng1, ZHU Xu-ying1, LI Jia-lin1, SHAO Xing-hua1, YANG Yuan-ting1, NI Zhao-hui1
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1.Department of Nephrology, Molecular Cell Lab for Kidney Disease, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Uremia Diagnosis and Treatment Center, Shanghai Jiao Tong University School of Medicine, Shanghai Peritoneal Dialysis Research Center, Shanghai 200127, China;2.Department of Nephrology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201200, China
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| Abstract: |
| Objective To explore the role of ferroptosis in LPS-induced sepsis associated acute kidney injury (S-AKI) via in vivo and in vitro models. Methods A mouse model of sepsis was established and randomly divided into 4 groups: the control group, lipopolysaccharide (LPS) group, LPS＋Ferrostatin-1 (Fer-1) group and LPS＋Deferoxamine (DFO) group, 3 mice for each group. Mice in LPS group were administered intraperitoneally with LPS at a dose of 15 mg/kg, and those in ferroptosis inhibitor group were intraperitoneally injected with Fer-1 (5 mg/kg) or DFO (100 mg/kg) 30 minutes before LPS injection, respectively. Blood and kidney samples were collected 24 hours later for renal function and ferroptosis markers detection. Human renal tubular epithelial cells (HK-2) were exposed to 100 μg/mL LPS stimulation for 24 h, and cell viability, labile iron pool (LIP) and lipid peroxidation were detected. After transfection to HK-2 cells with siRNA against ferroportin (FPN), the expression of ferroptosis markers were detected. Results Compared with the control group, the levels of serum creatinine and urea nitrogen of mice in LPS group were significantly higher, the renal pathological injury was aggravated, and ferroptosis markers were remarkably increased. However, ferroptosis inhibitors could significantly relieve the kidney functional and pathological injury induced by LPS, and reduce the expression of ferroptosis markers. Meanwhile, FPN expression was significantly down-regulated in LPS-induced S-AKI group both in vivo and in vitro, and obvious ferroptosis markers were observed in HK-2 cells silenced by siRNA for FPN, suggesting that FPN was involved in ferroptotic injury. qRT-PCR results showed that down-regulation of FPN was associated with increased hepcidin in the kidney. Further tests showed that LPS activated the hepcidin promoter IL-6/STAT3 pathway, while Fer-1 and DFO inhibited the activation of related pathway, reduced hepcidin mRNA expression, and prevented the down-regulation of FPN. Conclusions LPS can induce down-regulation of FPN mediated ferroptosis, and ferroptosis inhibitors may play a protective role in LPS-induced S-AKI through inhibiting the degradation of FPN by hepcidin. |
| Key words: ferroptosis sepsis acute kidney injury ferroportin hepcidin |
