血红蛋白诱导心肌细胞铁死亡及Basigin的调控机制研究

doi:10.3969/j.issn.1674-8115.2025.12.001

摘要/Abstract

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、30.0 μmol/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、7.5 μmol/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。

关键词: 心肌内出血, 缺血再灌注损伤, 血红蛋白, 心肌细胞, 铁死亡, Basigin

Abstract:

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 30.0 μmol/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 7.5 μmol/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 Hb-induced 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 concentration-dependent 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 prostaglandin-endoperoxide 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.

Key words: intramyocardial hemorrhage, ischemia-reperfusion injury, hemoglobin (Hb), cardiomyocyte, ferroptosis, Basigin (BSG)

中图分类号:

R542.2

李文丽, 钟方元, 赵怡超, 金力行, 雷杰, 石瑶, 卜军, 葛恒. 血红蛋白诱导心肌细胞铁死亡及Basigin的调控机制研究[J]. 上海交通大学学报（医学版）, 2025, 45(12): 1559-1567.

LI Wenli, ZHONG Fangyuan, ZHAO Yichao, JIN Lixing, LEI Jie, SHI Yao, PU Jun, GE Heng. Mechanisms of Basigin regulation in hemoglobin-induced cardiomyocyte ferroptosis[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(12): 1559-1567.

图/表 7

表1 siRNA序列（5′→3′）

Tab 1 Sequences of siRNA （5'→3'）

siRNA	Forward	Reverse
si-NC	UUCUCCGAACGUGUCACGUdTdT	ACGUGACACGUUCGGAGAAdTdT
si-Bsg	GAUCAAGGUGGGAAAGAAtt	UUUCUUUCCCACCUUGAUCtt

表2 实时荧光定量PCR引物序列（5′→3′）

Tab 2 Primer sequences for real-time quantitative PCR （5′→3′）

Gene	Forward primer	Reverse primer
Ptgs2	TTCCTCCTGTGGCTGATGACTG	AGGTCCTCGCTTCTGATCTGTC
Bsg	GCATCTTCCTTCCTGAGCCTGTG	TGGCGTGTTCCGATTTCTTTCCC
β-actin	CACTATCGGCAATGAGCGGTTC	CAGCACTGTGTTGGCATAGAGG

图1 Hb诱导H9c2心肌细胞损伤且呈浓度依赖性Note： A. Flow cytometry analysis of PI-positive H9c2 cardiomyocytes after treatment with different concentrations of Hb for 24 h. B. Quantitative analysis of PI-positive rates in H9c2 cardiomyocytes. C. WST-1 assay to detect the viability of H9c2 cardiomyocytes after treatment with different concentrations of Hb. ①P=0.044, ②P<0.001, ③P=0.020.

Fig 1 H9c2 cardiomyocyte injury induced by Hb in a concentration-dependent manner

图2 Hb加重缺氧诱导的H9c2心肌细胞损伤Note： A. Flow cytometry analysis of PI-positive H9c2 cardiomyocytes after hypoxia (1% O₂, 6 h) followed by treatment with different concentrations of Hb for 24 h. B. Quantitative analysis of PI-positive rates in H9c2 cardiomyocytes. C. WST-1 assay to detect the viability of H9c2 cardiomyocytes after hypoxia followed by treatment with different concentrations of Hb. ①P=0.032, ②P<0.001, ③P=0.042, ④P=0.010.

Fig 2 Hypoxia-induced H9c2 cardiomyocyte injury exacerbated by Hb

图3 铁死亡抑制剂对Hb诱导的H9c2心肌细胞损伤的影响Note： A. WST-1 assay to detect the effects of different cell death inhibitors on the viability of H9c2 cardiomyocytes induced by Hb. B. Flow cytometry analysis of the effect of Fer-1 on PI-positive rate in Hb-treated H9c2 cardiomyocytes. C. Quantitative analysis of the flow cytometry results. ①P<0.001.

Fig 3 Effect of the ferroptosis inhibitor on Hb-induced H9c2 cardiomyocyte injury

图4 Hb诱导的H9c2心肌细胞中BSG表达变化Note： A. Western blotting analysis of BSG protein expression levels in two groups of H9c2 cardiomyocytes. B. Real-time quantitative PCR analysis of Bsg mRNA expression levels in two groups of H9c2 cardiomyocytes. ①P<0.001.

Fig 4 Hb-induced changes in BSG expression in H9c2 cardiomyocytes

图5 敲低 Bsg 对Hb诱导的H9c2心肌细胞损伤的影响Note： A. Western blotting analysis of BSG protein expression levels after si-NC or si-Bsg transfection. B. Real-time quantitative PCR analysis of Bsg mRNA expression levels after si-NC or si-Bsg transfection. C. Flow cytometry analysis of the effect of Bsg silencing on the PI-positive rate in Hb-treated H9c2 cardiomyocytes. D. Quantitative analysis of the flow cytometry results. E. WST-1 assay to detect the viability of H9c2 cardiomyocytes in the indicated groups. F. Real-time quantitative PCR analysis of Ptgs2 mRNA expression in Hb-treated H9c2 cardiomyocytes after Bsg silencing. ①P<0.001, ②P=0.011.

Fig 5 Effects of knocking down Bsg on Hb-induced H9c2 cardiomyocyte injury

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