简介:由于吡柔比星(THP)的心脏毒性,有必要研究用于治疗THP引起的心脏毒性的新化合物。异槲皮苷(IQC)是一种具有抗氧化和抗凋亡特性的天然类黄酮。因此,本研究旨在研究IQC在体内和体外预防THP诱导的心脏毒性的影响。方法:通过MTT法测定IQC预防THP诱导的心肌细胞损伤所需的最佳浓度和时间。使用二氯二氢荧光素二乙酸荧光探针在H9c2和HCM细胞中进一步验证了保护作用,MitoTracker红色探测器,酶联免疫吸附测定,JC-1探头,和实时定量聚合酶链反应(RT-qPCR)。给大鼠施用THP以建立心脏毒性。做了心电图(ECG),和心脏血流动力学,心肌酶,氧化应激指标,并对苏木精-伊红染色进行了研究。电压依赖性阴离子通道1(VDAC1),腺嘌呤核苷酸转位酶1(ANT1),qRT-PCR检测亲环蛋白D(CYPD),Westernblot检测Phlpp1/AKT/Bcl-2轴蛋白,证实IQC显着增加细胞活力和超氧化物歧化酶(SOD)水平,降低了ROS和MDA的水平,以及体内和体外线粒体功能和细胞凋亡的升高。结果:结果表明,IQC减轻了THP诱导的心肌组织病理学损伤,心电图(ECG)异常,和体内心功能不全。IQC还降低血清MDA水平,BNP,CK-MB,c-TnT,LDH,同时增加SOD和GSH的水平。我们还发现IQC显着降低VDAC1,ANT1和CYPDmRNA的表达。此外,IQC通过调节Phlpp1/AKT/Bcl-2信号通路控制细胞凋亡。IQC显着增加H9c2和HCM细胞活力和SOD水平,降低了ROS和MDA的水平,H9c2和HCM细胞线粒体功能增强,以防御THP诱导的心肌细胞凋亡。AKT抑制剂IMQ证明IQC缺乏抗氧化和抗凋亡特性。此外,我们的数据显示IQC调节Phlpp1表达,从而影响p-AKT的表达水平,细胞色素c,caspase-3、caspase-9、Bcl-2和Bax。讨论:总之,我们的结果表明,IQC通过调节Phlpp1/AKT/Bcl-2信号通路保护心肌细胞线粒体膜通透性的变化,抑制细胞从线粒体内膜释放到细胞质,形成凋亡体,诱导细胞凋亡,并减少THP诱导的心脏毒性。
Introduction: Due to the cardiotoxicity of
pirarubicin (THP), it is necessary to investigate new compounds for the treatment of THP-induced cardiotoxicity. Isoquercitrin (IQC) is a natural flavonoid with anti-oxidant and anti-apoptosis properties. Thus, the present study aimed to investigate the influence of IQC on preventing the THP-induced cardiotoxicity in vivo and in vitro. Methods: The optimal concentration and time required for IQC to prevent THP-induced cardiomyocyte damage were determined by an MTT assay. The protective effect was further verified in H9c2 and HCM cells using dichlorodihydrofluorescein diacetate fluorescent probes, MitoTracker Red probe, enzyme-linked immunosorbent assay, JC-1 probe, and real time-quantitative polymerase chain reaction (RT-qPCR). Rats were administered THP to establish cardiotoxicity. An electrocardiogram (ECG) was performed, and cardiac hemodynamics, myocardial enzymes, oxidative stress indicators, and hematoxylin-eosin staining were studied. Voltage-dependent anion channel 1 (VDAC1), adenine nucleotide translocase 1 (ANT1), and cyclophilin D (CYPD) were detected by qRT-PCR, and the Phlpp1/AKT/Bcl-2 axis proteins were detected by western blot, confirming that IQC markedly increased cell viability and superoxide dismutase (SOD) levels, diminished the levels of ROS and MDA, and elevated mitochondrial function and apoptosis in vivo and in vitro. Results: Results showed that IQC reduced THP-induced myocardial histopathological injury, electrocardiogram (ECG) abnormalities, and cardiac dysfunction in vivo. IQC also decreased serum levels of MDA, BNP, CK-MB, c-TnT, and LDH, while increasing levels of SOD and GSH. We also found that IQC significantly reduced VDAC1, ANT1, and CYPD mRNA expression. In addition, IQC controlled apoptosis by modulating Phlpp1/AKT/Bcl-2 signaling pathways. IQC markedly increased H9c2 and HCM cell viability and SOD levels, diminished the levels of ROS and MDA, and elevated mitochondrial function in H9c2 and HCM cells to defend against THP-induced cardiomyocyte apoptosis in vitro. The AKT inhibitor IMQ demonstrated that IQC lacked antioxidant and anti-apoptotic properties. Moreover, our data showed that IQC regulates Phlpp1 expression, thereby influencing the expression levels of p-AKT, cytochrome c, caspase-3, caspase-9, Bcl-2, and Bax. Discussion: In conclusion, our results indicate that IQC protects the changes in mitochondrial membrane permeability in cardiomyocytes by regulating the Phlpp1/AKT/Bcl-2 signaling pathway, inhibits the release of cytc from the mitochondrial inner membrane to the cytoplasm, forms apoptotic bodies, induces cell apoptosis, and reduces THP induced cardiotoxicity.