Qishen Granule Attenuates Doxorubicin-induced Cardiotoxicity by Protecting Mitochondrial Function and Reducing Oxidative Stress Through Regulation of Sirtuin3

Ethnopharmacological Relevance: Doxorubicin (DOX) is one of the most potent chemotherapy drugs available today. However, the adverse effect of cardiotoxicity limits its clinical application. New approaches are being investigated for the treatment of doxorubicin-induced cardiotoxicity (DIC). Doxorubicin is enriched in mitochondria and it could induce imbalance of protein modification, including acetylation of mitochondria proteins, thereby inducing DIC. Restoration of mitochondria function is an effective way to attenuate DIC. The formula for traditional Chinese medicine Granules of Qishen (QSG) was derived from the classic formula "Zhen-Wu-Tang" which has been extensively used in the treatment of myocardial infarction. It consists of six traditional Chinese medicines, including Astragalus membranaceus var. mongholicus (Bunge) P.K.Hsiao (Fabaceae), Salvia miltiorrhiza Bunge (Lamiaceae), Lonicera japonica Thunb. (Caprifoliaceae), Aconitum carmichaelii Debeaux (Ranunculaceae), Scrophularia ningpoensis Hemsl. (Scrophulariaceae), and Glycyrrhiza uralensis Fisch. (Fabaceae). QSG is a potential anti-DIC formula. A better understanding of the effectiveness and pharmacological mechanisms of QSG will aid in the prevention and treatment of DIC.

Aim Of The Study: The purpose of this research was to explore the effectiveness of QSG in the treatment of DIC and to explore whether QSG could protect mitochondrial function and reduce oxidative damage by activating Sirtuin3(SIRT3)/Acetylated-superoxide dismutase 2(Ac-SOD2) signaling pathway.

Materials And Methods: DOX was injected into mice through the tail vein to construct a mouse model of DOX-induced cardiotoxicity to explore the therapeutic effect of QSG in animals. Meanwhile, the H9C2 cell model was used to study the mechanism of QSG. The cardiac function was evaluated by echocardiography, hematoxylin-eosin (H&E) staining and measurement of serum levels of creatine kinase isoenzymes (CK-MB) and lactate dehydrogenase (LDH). Oxidative damage was evaluated by 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA) staining and Mito-SOX Red staining. Levels of total superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by following the instructions of commercially available kits. In order to detect the changes in mitochondrial membrane potential, cells were stained using the mitochondrial membrane potential detection kit (JC-1). Western blot analysis was applied to detect protein expressions of SIRT3, Ac-SOD2, Acetylation Lysine (Ac-Lys), Bax and Bcl-2. H9C2 cells were treated with SIRT3 inhibitor, in order to determine if QSG had effects via the SIRT3/Ac-SOD2 pathway.

Results: In vivo studies showed that QSG ameliorated doxorubicin-induced damage of cardiac function in DIC mice model. The ejection fraction (EF) and fractional shortening (FS) were all up-regulated by QSG treatment. QSG decreased MDA levels and increased SOD activity. Meanwhile, doxorubicin induced high level of protein acetylation and QSG restored the acetylated protein back to normal levels. In particular, QSG upregulated expression of SIRT3 and downregulated Ac-SOD level. In vitro study demonstrated that QSG restored mitochondrial membrane potential, increased ATP level and reduced mitochondrial ROS production. When H9C2 cells were co-incubated with SIRT3 inhibitor, the efficacies of QSG on mitochondrial function were abrogated. Meanwhile, the regulative effects of QSG on SIRT3/Ac-SOD2 pathway were also abolished.

Conclusion: This study demonstrates that QSG is effective in treating DIC. QSG ameliorates oxidative damage and protects mitochondrial function partly by restoring protein acetylation level and by activating the SIRT3/Ac-SOD2 pathway.