Quercetin Regulates Mitochondrial Damage and Ferroptosis in Sepsis-induced Cardiomyopathy in Mice and Cardiomyocytes via TFRC
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摘要:
目的 探讨槲皮素（quercetin，QUE）调节转铁蛋白受体（transferrin receptor，TFRC）对脓毒症心肌病模型细胞线粒体和铁死亡的影响并阐明相关调节机制。 方法 利用1 μg/mL脂多糖（lipopolysaccharide，LPS）分别与20 μM、40 μM、80 μM、160 μM QUE培养H9C2细胞24 h。在80 μM QUE处理的H9C2细胞中转染sh-TFRC、pcDNA-TFRC或用铁死亡抑制剂Ferrostatin-1处理24 h。通过CCK-8、流式细胞术、JC-1染色、Western blot、试剂盒、酶联免疫吸附和RT-qPCR实验探讨QUE对TFRC表达的调控以及QUE调控TFRC对LPS诱导H9C2细胞线粒体功能和铁死亡的影响。QUE经腹腔注射至小鼠并经LPS诱导SCM小鼠模型，经苏木精-伊红染色、TUNEL染色、ELISA和试剂盒以及Western blot实验探讨QUE对SCM小鼠的影响。 结果 LPS诱导可抑制H9C2细胞增殖，促进细胞凋亡、ROS、Cleaved-caspase 3、Bax、FTH1、ACSL4、Fe2+、白介素-1β（interleukin-1β，IL-1β、肿瘤坏死因子（tumor necrosis factor-α，TNF-α）、乳酸脱氢酶（lactate dehydrogenase，LDH）和肌酸激酶同工酶MB（creatine kinase-MB，CK-MB）水平（P < 0.001），降低线粒体膜电位和Bcl-2、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）蛋白表达（P < 0.001）；经40 μM、80 μM、160 μM QUE处理可减轻LPS诱导的H9C2细胞线粒体功能障碍和铁死亡（均P < 0.05），抑制TFRC表达（P < 0.001）；敲低TFRC或经Ferrostatin-1处理可进一步增强80 μM QUE对H9C2细胞损伤的保护作用（均P < 0.05）。过表达TFRC可增强LPS诱导的H9C2细胞线粒体损伤和铁死亡，QUE处理可缓解过表达TFRC对H9C2的作用。LPS诱导的SCM小鼠心肌细胞损伤和死亡增加、炎性细胞因子水平增加；经QUE处理的SCM小鼠心肌损伤缓解，铁死亡被抑制。 结论 QUE通过下调TFRC表达，抑制铁死亡，缓解LPS诱导的H9C2细胞线粒体损伤以及小鼠心肌损伤。 Abstract:Objective To investigate the effects of quercetin （QUE） on regulating transferrin receptor （TFRC） expression on mitochondrial function and ferroptosis in a sepsis-induced cardiomyopathy （SCM） cell model, and to elucidate the underlying regulatory mechanisms. Methods H9C2 cells were treated with 1 μg/mL LPS and co-cultured with 20 μM, 40 μM, 80 μM, or 160 μM QUE for 24 hours. H9C2 Cells treated with 80 μM QUE were transfected with sh-TFRC, pcDNA-TFRC, or treated with the ferroptosis inhibitor Ferrostatin-1 for 24 hours. The regulatory effect of QUE on TFRC expression and the effects of QUE-regulated TFRC regulation on LPS-induced mitochondrial dysfunction and ferroptosis in H9C2 cells were assessed using CCK-8 assay, flow cytometry, JC-1 staining, Western blot, commercial kits, enzyme-linked immunosorbent assay （ELISA）, and RT-qPCR. QUE was intraperitoneally injected into mice and LPS was administered to establish an SCM mouse model. The effects of QUE on SCM mice were investigated using hematoxylin-eosin staining, TUNEL staining, ELISA, commercial kits, and Western blotting. Results LPS stimulation significantly inhibited H9C2 cell proliferation, promoted cell apoptosis, and increased the levels of ROS, cleaved-caspase 3, Bax, FTH1, ACSL4, Fe2+, IL-1β, TNF-α, LDH, and CK-MB （P < 0.001）, while decreasing mitochondrial membrane potential and expression of Bcl-2 and glutathione peroxidase 4 （GPX4） proteins （P < 0.001）. Treatment with 40 μM, 80 μM, and 160 μM QUE significantly alleviated LPS-induced mitochondrial dysfunction and ferroptosis in H9C2 cells （all P < 0.05） and inhibited TFRC expression （P < 0.001）. TFRC knockdown or Ferrostatin-1 treatment further enhanced the protective effect of 80 μM QUE against H9C2 cell injury （all P < 0.05）. TFRC overexpression exacerbated LPS-induced mitochondrial dysfunction and ferroptosis in H9C2 cells, while QUE treatment alleviated the effects of TFRC overexpression on H9C2 cells. LPS stimulation increased cardiac myocyte injury and death and elevated inflammatory cytokines levels in SCM mice. QUE treatment mitigated myocardial injury and suppressed ferroptosis in SCM mice. Conclusion QUE alleviates LPS-induced mitochondrial injury in H9C2 cells and cardiac myocardial injury in mice by downregulating TFRC expression and inhibiting ferroptosis. -
Key words:
- Septic cardiomyopathy /
- Quercetin /
- TFRC /
- Mitochondria /
- Ferroptosis
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图 1 QUE对H9C2细胞增殖、凋亡和ROS的调节作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A：CCK-8检测细胞增殖；B～C：流式细胞术检测细胞凋亡率；D～E：流式细胞术检测细胞中的ROS；*P<0.05；**P<0.01；***P<0.001。
Figure 1. Regulatory effects of QUE on H9C2 cell proliferation，apoptosis，and ROS （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 2 QUE对H9C2细胞线粒体膜电位、凋亡蛋白和Fe2+的调节作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A～B：JC-1染色检测细胞线粒体膜电位；C～F：Western blot检测细胞凋亡相关蛋白Cleaved-caspase 3、Bax和Bcl-2蛋白表达；G：试剂盒检测细胞中Fe2+的浓度；**P<0.01；***P<0.001。
Figure 2. Regulatory effects of QUE on mitochondrial membrane potential，apoptotic proteins，and Fe2+ in H9C2 cells （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 3 QUE对H9C2细胞铁死亡、细胞因子、LDH和CK-MB的调节作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A～D：Western blot检测QUE对铁死亡相关标记物GPX4、FTH1、ACSL4蛋白表达的作用；E～F：ELISA试剂盒检测细胞中IL-1β和TNF-α的浓度；G～H：生化试剂盒检测细胞中LDH和MDA浓度；I：ELISA试剂盒检测细胞中CK-MB的浓度；**P<0.01；***P<0.001。
Figure 3. Regulatory effects of QUE on ferroptosis，cytokines，LDH，and CK-MB in H9C2 cells （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 4 QUE下调LPS诱导的H9C2细胞中TFRC表达（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A：RT-qPCR检测TFRC mRNA相对表达；B～C：Western blot检测TFRC蛋白相对表达；***P<0.001。
Figure 4. QUE downregulates TFRC expression in LPS-induced H9C2 cells （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 5 敲低TFRC增强QUE对H9C2细胞增殖、凋亡和ROS的作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A～B：Western blot检测转染sh-TFRC细胞中TFRC蛋白相对表达；C：RT-qPCR检测sh-TFRC细胞中TFRC mRNA相对表达；D：CCK-8检测细胞增殖；E和G：流式细胞术检测细胞凋亡率；F和H：流式细胞术检测细胞中ROS水平；*P<0.05；**P<0.01；***P<0.001。
Figure 5. Effects of TFRC knockdown on QUE-enhanced proliferation，apoptosis，and ROS in H9C2 cells（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 6 敲低TFRC增强QUE对H9C2细胞线粒体膜电位、凋亡蛋白和Fe2+的作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A～B：JC-1染色评估H9C2细胞线粒体膜电位；C～F：Western blot检测细胞中凋亡相关蛋白的相对表达；G：试剂盒检测细胞中Fe2+的浓度；*P<0.05；**P<0.01；***P<0.001。
Figure 6. Effects of TFRC knockdown on QUE-enhanced mitochondrial membrane potential，apoptotic proteins，and Fe2+ in H9C2 cells （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 7 敲低TFRC增强QUE对H9C2细胞铁死亡和细胞因子的调节作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A～D：Western blot检测铁死亡相关标记物GPX4、FTH1、ACSL4蛋白表达；E～F：ELISA试剂盒检测敲低TFRC对H9C2细胞培养上清中TNF-α和IL-1β水平的作用；G～H：生化试剂盒检测LDH水平；I：ELISA试剂盒检测细胞上清中CK-MB水平；**P<0.01；***P<0.001。
Figure 7. Knockdown of TFRC enhances the regulatory effects of QUE on ferroptosis and cytokines in H9C2 cells （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 8 QUE通过下调TFRC缓解LPS诱导对H9C2细胞活性、凋亡和ROS的作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A～B：Western blot检测转染pcDNA-TFRC和QUE处理组中TFRC蛋白表达；C：RT-qPCR检测TFRC mRNA相对表达；D：CCK-8检测过表达TFRC对细胞活力的影响；E和G：Annexin V-FITC/PI试剂盒检测细胞凋亡率；F和H：DCFH-DA探针检测细胞中的ROS水平；*P<0.05；**P<0.01；***P<0.001。
Figure 8. QUE alleviates LPS-induced effects on H9C2 cell viability，apoptosis，and ROS by downregulating TFRC （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 9 QUE通过下调TFRC缓解LPS诱导对H9C2细胞线粒体膜电位、凋亡蛋白和Fe2+的作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A～B：JC-1染色检测过表达TFRC对H9C2细胞线粒体膜电位的影响；C～F：Western blot检测过表达TFRC对细胞中的Cleaved-caspase 3、Bax、Bcl-2蛋白表达的作用；G：生化试剂盒检测细胞中的Fe2+浓度；*P<0.05；**P<0.01；***P<0.001。
Figure 9. QUE alleviates the effects of LPS induction on mitochondrial membrane potential，apoptosis proteins，and Fe2+ in H9C2 cells by downregulating TFRC （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 10 QUE通过下调TFRC缓解LPS诱导对H9C2细胞铁死亡和炎症反应的作用（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
A～D：Western blot检测过表达TFRC对铁死亡相关蛋白GPX4、FTH1和ACSL4蛋白表达的作用；E～F：ELISA试剂盒检测细胞上清液中的TNF-α和IL-1β水平；G～H：生化试剂盒检测细胞中LDH和MDA浓度；I：ELISA试剂盒检测细胞中CK-MB的浓度；*P<0.05；**P<0.01；***P<0.001。
Figure 10. Quercetin alleviates LPS-induced ferroptosis and inflammatory response in H9C2 cells through downregulation of TFRC （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 3）
图 11 QUE通过下调TFRC缓解小鼠SCM（$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 6）
A：小鼠心肌组织HE染色（20×）；B：小鼠心肌组织TUNEL染色（40×）；C～D：ELISA试剂盒检测小鼠心肌组织中的TNF-α和IL-1β浓度；E～F：生化试剂盒检测小鼠心肌组织中的LDH和MDA；G：ELISA试剂盒检测心肌组织中的CK-MB浓度；H～L：Western blot检测心肌组织中的TFRC、GPX4、FTH1和ACSL4蛋白表达；*P<0.05；**P<0.01；***P<0.001。
Figure 11. QUE alleviates SCM in mice by downregulating TFRC （$ \overline{\boldsymbol{x}} \pm \boldsymbol{s} $，n = 6）
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