Abstract
Doxorubicin (DOX) is a widely used antineoplastic agent in clinics. However, its clinical application is largely limited by its cardiotoxicity. Diethyl blechnic (DB) is a novel compound isolated from Salvia miltiorrhiza Bunge. Here, we study the effect of DB on DOX-induced cardiotoxicity and its underlying mechanisms. Cellular viability was tested by 3-[-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and protein level was evaluated by Western blotting. 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining was performed to determine the mitochondrial membrane potential (MMP). Hoechst 33342 staining and TUNEL staining was performed to test the apoptosis. Reactive oxygen species (ROS) generation was investigated by using flow cytometry. DB significantly inhibited DOX-induced apoptosis in H9c2 cells and primary cultured cardiomyocytes. Moreover, DB decreased cell apoptotic morphological changes and reversed the mitochondrial membrane potential induced by DOX. Meanwhile, pre-treatment with DB increased the expression levels of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xl), and survivin and reduced the expression levels of Bcl-2-associated X protein (Bax), p-p53, cytochrome c (cyt c), and cleaved-caspase 3, 7, 8, 9 in the protein levels in DOX-treated H9c2 cells. Furthermore, DB suppressed ROS generation. The DB-mediated protective effects were accompanied by increased c-Jun N-terminal kinase1/2 (JNK1/2) expression. In addition, SP600125, the inhibitor of JNK1/2, abolished the protective effect of DB. We concluded that DB protected cardiomyocytes against DOX-induced cytotoxicity by inhibiting ROS and activating the JNK1/2 pathway. Therefore, DB is a promising candidate as a cardioprotective agent against DOX-induced cardiotoxicity.
Highlights
Doxorubicin (DOX), the representative anticancer anthracycline in chemotherapy, was first isolated from Streptomyces peucetius in the 1960s [1]
We demonstrated that Diethyl blechnic (DB), which is a novel compound isolated from Danshen, can protect against DOX-induced cardiotoxicity in vitro
The H9c2 cell is the most popular in vitro cell model used for cardiovascular research, for DOX-induced cytotoxicity [19,20] and it has some different characteristics from primary cardiomyocytes
Summary
Doxorubicin (DOX), the representative anticancer anthracycline in chemotherapy, was first isolated from Streptomyces peucetius in the 1960s [1]. DB Protects from DOX-Induced Cardiac Cell Apoptosis. The total cellular ROS levels were measured by using flow cytometry. The results showed that H9c2 cells dOisxpidlaayteidveosbtvrieosussliyn hHig9hce2r cReOllSs bacycupmrouvlaotkioinngafitnertrDacOeXllutlraeratRmOenSt gcoemnepraarteidonwiisthththeemcoonsttrowl idely accepted mechanism for DOX cardiotoxicity. We determined the ROS levels to examine whether DB protected against DOX-induced toxicity in H9c2 cells by inhibiting ROS. Obviously higher ROS accumulation after DOX treatment compared with the control group, whereas DB pIrnet.-Jt.rMeoal.tmScie. Group, whereas DB pre-treatment clearly inhibited ROS accumulation (Figure 5). These results indicate5dotfh1a3 t DB exerted its protective effects by inhibiting ROS.
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