摘要
目的·探讨血红蛋白(hemoglobin,Hb)诱导心肌细胞损伤的机制,以及Basigin(BSG)在其中的调控作用。方法·构建体外实验模型,采用不同浓度Hb(0、7.5、15.0、30.0μmol/L)处理H9c2心肌细胞,并利用WST-1法和流式细胞术检测细胞活性及死亡率;随后,对H9c2心肌细胞进行缺氧/复氧处理,并加入低浓度梯度的Hb(0、2.5、5.0、7.5μmol/L)模拟缺血再灌注损伤的病理微环境,以进一步验证Hb对心肌细胞的毒性作用。使用多种细胞死亡抑制剂,包括坏死性凋亡抑制剂(necrostatin-1,Nec-1)、自噬抑制剂(3-methyladenine,3-MA)、铁死亡抑制剂(ferrostatin-1,Fer-1)、焦亡抑制剂(VX-765)干预,以探究Hb促进心肌细胞损伤的机制。采用Western blotting及实时荧光定量PCR检测Hb诱导后心肌细胞中Bsg mRNA和蛋白质表达变化。采用siRNA敲低H9c2心肌细胞中Bsg的表达水平,并通过WST-1法和流式细胞术验证BSG在Hb诱导的心肌细胞损伤和铁死亡过程中的作用。结果·无论在常氧还是缺氧/复氧条件下,Hb均对H9c2心肌细胞表现出直接的毒性作用,且该毒性作用呈现浓度依赖性。进一步研究发现,相较于其他细胞死亡抑制剂,铁死亡抑制剂Fer-1能够更显著地减轻Hb诱导的心肌细胞损伤。Western blotting和实时荧光定量PCR结果显示,与对照组相比,Hb处理组H9c2心肌细胞中Bsg的mRNA和蛋白表达水平显著增加。敲低Bsg的表达能够降低铁死亡标志物前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,Ptgs2) mRNA的表达,并减轻Hb诱导的心肌细胞损伤和死亡。结论·Hb可能通过诱导心肌细胞铁死亡导致心肌损伤;BSG在此过程中发挥一定作用,抑制其表达能够抵抗Hb诱导的铁死亡和心肌细胞损伤。
Objective·To investigate the mechanism of hemoglobin(Hb)-induced cardiomyocyte injury and the regulatory role of Basigin(BSG)in this process.Methods·An in vitro model was established by treating H9c2 cardiomyocytes with different concentrations of Hb(0,7.5,15.0,and 30.0μmol/L);cell viability and mortality were detected using the WST-1 assay and flow cytometry.Subsequently,H9c2 cardiomyocytes underwent hypoxia/reoxygenation treatment with low-concentration gradients of Hb(0,2.5,5.0,and 7.5μmol/L)to simulate the pathological microenvironment of ischemia-reperfusion injury,further validating Hb's toxic effects on cardiomyocytes.Multiple cell death inhibitors were used,including a necroptosis inhibitor(necrostatin-1,Nec-1),an autophagy inhibitor(3-methyladenine,3-MA),a ferroptosis inhibitor(ferrostatin-1,Fer-1),and a pyroptosis inhibitor(VX-765),to investigate the mechanism of Hb-induced cardiomyocyte injury.Bsg mRNA and protein levels were detected by Western blotting and real-time quantitative PCR.Bsg expression was knocked down in H9c2 cardiomyocytes using siRNA;the role of BSG in Hbinduced cardiomyocyte injury and ferroptosis was then verified by the WST-1 assay and flow cytometry.Results·Under both normoxic and hypoxia/reoxygenation conditions,Hb showed direct toxic effects on H9c2 cardiomyocytes in a concentrationdependent manner.Further investigation showed that,compared with other cell death inhibitors,the ferroptosis inhibitor Fer-1 more significantly alleviated Hb-induced cardiomyocyte injury.Western blotting and real-time quantitative PCR results demonstrated that compared with the control group,Bsg mRNA and protein expression levels were significantly increased in Hb-treated H9c2 cardiomyocytes.Knockdown of Bsg expression decreased the mRNA expression of the ferroptosis marker prostaglandinendoperoxide synthase 2(Ptgs2)and alleviated Hb-induced cardiomyocyte injury and death.Conclusion·Hb may induce myocardial injury by promoting cardiomyocyte ferroptosis;BSG plays a role in this process,and inhibition of its expression can counteract Hb-induced ferroptosis and cardiomyocyte injury.
作者
李文丽
钟方元
赵怡超
金力行
雷杰
石瑶
卜军
葛恒
LI Wenli;ZHONG Fangyuan;ZHAO Yichao;JIN Lixing;LEI Jie;SHI Yao;PU Jun;GE Heng(Department of Cardiology,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China)
出处
《上海交通大学学报(医学版)》
北大核心
2025年第12期1559-1567,共9页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(81770238)
新疆维吾尔自治区自然科学基金(2022D01C16)
上海市卫生健康委员会学科带头人计划(2022XD018)。
