摘要
目的探讨Necrostatin-1(Nec-1)对免疫检查点抑制剂相关心肌炎(immune checkpoint inhibitor-associated myocarditis,ICIAM)小鼠模型病情的改善作用及潜在机制。方法选择10只6~8周雄性BALB/c小鼠,建立ICIAM小鼠模型。采用超声心动图和血清心肌损伤标志物评估小鼠心功能。分别采用酶联免疫吸附试验(ELISA)和实时荧光定量聚合酶链式反应(qRT-PCR)检测炎症标志物肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)水平。通过苏木精-伊红(HE)染色评估心肌组织炎症细胞浸润水平;通过Masson染色评估心肌纤维化程度。采用Western印迹法检测小鼠心肌坏死性凋亡相关蛋白,包括受体相互作用蛋白1(RIP1)、RIP3、混合谱系激酶结构域样蛋白(MLKL)及其磷酸化形式的表达。提取脾脏淋巴细胞,将其与HL-1细胞系共培养,采用CCK-8法检测细胞活力,观察HL-1细胞中线粒体活性氧(ROS)释放和线粒体膜电位变化,测定HL-1细胞内RIP1、RIP3、MLKL及其磷酸化形式表达。检测HL-1细胞中氧化应激标志物丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性。结果Nec-1能改善ICI导致的小鼠心功能损害,减少ICIAM小鼠血浆中TNF-α和IL-1β的水平(P<0.001);抑制磷酸化RIP1、RIP3和MLKL的表达(P<0.05);降低MDA活性,提高SOD活性和GSH-Px活性(P<0.001)。HL-1细胞中,Nec-1干预能抑制RIP1-RIP3-MLKL通路激活(P<0.05);改善由淋巴细胞诱导的HL-1细胞活力降低(P<0.001);减少线粒体ROS释放,增加线粒体膜电位,降低MDA活性,提高SOD和GSH-Px活性(P<0.001)。结论坏死性凋亡在ICIAM的发生发展中起重要作用,而Nec-1可通过抑制心肌细胞氧化应激减少坏死性凋亡,缓解ICIAM进展;RIP1可能为未来ICIAM治疗的新靶点。
Objective To investigate the improvement effect of Necrostatin-1(Nec-1)on mouse models with immune checkpoint inhibitor(ICI)-associated myocarditis(ICIAM)and potential mechanism.Methods Ten male BALB/c mice aged 6-8 weeks were selected to construct the ICIAM models.The echocardiography and serum myocardial injury markers were used to assess cardiac function of mice.The levels of inflammatory markers including tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by enzyme-linked immunosorbent assay(ELISA)and quantitative real-time polymerase chain reaction(qRT-PCR),respectively.Hematoxylin-eosin(HE)staining was used to evaluate myocardial inflammation,and Masson staining was used to evaluate myocardial fibrosis.The expressions of myocardial necroptosis proteins including receptor-interacting protein kinase 1(RIP1),RIP3,mixed lineage kinase domain-like protein(MLKL)and their phosphorylated forms were detected by Western blotting.The spleen lymphocytes were extracted and co-cultured with HL-1 cell line.Cell viability was measured by cell counting kit-8(CCK-8).The release of reactive oxygen species(ROS)and changes of mitochondrial membrane potential were observed.RIP1,RIP3,MLKL and their phosphorylated forms were determined.The levels of markers of oxidative stress,including malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),were measured.Results Nec-1 significantly improved the cardiac function injury of mice induced by ICI,and inhibited the release of TNF-αand IL-1βin plasma of ICIAM mice(P<0.001);inhibited expressions of phosphorylated RIP1,RIP3 and MLKL(P<0.05);decreased MDA activity,and increased SOD and GSH-Px activity(P<0.001).In HL-1 cells,Nec-1 intervention inhibited the RIP1-RIP3-MLKL pathway(P<0.05),improved decrease of the cell viability induced by lymphocytes(P<0.001),decreased ROS release,increased mitochondrial membrane potential,inhibited MDA activity,and increased SOD and GSH-Px activities(P<0.001).Conclusions Necroptosis plays an important role in the occurrence and development of ICIAM,but Nec-1 could alleviate the progression of ICIAM by inhibiting necroptosis induced by oxidative stress in cardiomyocytes;RIP1 maybe a new target in treatment of ICIAM.
作者
汪雪君
沈毅辉
何小珍
张健
王妍
程蕾蕾
WANG Xuejun;SHEN Yihui;HE Xiaozhen;ZHANG Jian;WANG Yan;CHENG Leilei(Department of Cardiology,Zhongshan Hospital,Fudan University,Shanghai 200032,China;Immunotherapy Technology Transformation Research Center,Zhongshan Hospital,Fudan University,Shanghai 200032,China;Department of Cardiac Ultrasound Diagnosis,Zhongshan Hospital,Fudan University,Shanghai 200032,China;Department of Oncology,Zhongshan Hospital,Fudan University,Shanghai 200032,China)
出处
《中国临床医学》
2025年第6期1000-1009,共10页
Chinese Journal of Clinical Medicine
基金
国家自然科学基金(82170359,82470354)
非传染性慢性病国家科技重大专项(2023ZD0502800)
中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2024-JKCS-28)
上海市启明星培育(扬帆专项,24YF2744200)
上海市申康医院发展中心市级医院诊疗技术项目(SHDC22023207)。
