Mechanism of Sini Decoction and its self-assembled component in improving Doxorubicin-induced cardiomyopathy.

ETHOPHARMACOLOGICAL RELEVANCE: Sini decoction (SND) is a typical Traditional Chinese Medicine (TCM) prescription composed of Aconitum carmichaelii Debx, Zingiberis Rhizoma, and Glycyrrhizae Radix et Rhizoma Praeparate cum Melle, used to treat cardiomyopathy in TCM clinical trials. Recent studies have shown that the active ingredients of SND can form self-assembled complexes during decoction, whose bioactivity differs from that of the monomeric compound. However, current research on the active ingredients of SND has not yet included these self-assembled components, and the mechanism by which SND exerts its effects on doxorubicin (Dox)-induced cardiomyopathy remains unclear. AIM OF THE STUDY: This study aimed to investigate the underlying mechanisms by which SND and its self-assembled component (SAC) improve Dox-induced cardiomyopathy in mice. The complete isolation of the self-assembled complex component from SND was achieved for the first time, and its activity and mechanism were explored, providing a new perspective for revealing the substance basis of TCM prescriptions. MATERIALS AND METHODS: SAC and the unassembled component (SMC) were separated from SND by ultrafiltration, and UHPLC-Q-TOF-MS/MS was used to qualitatively analyze their chemical components. Based on the main components of SAC, Benzoylmesaconine-glycyrrhizic acid complex (GBMA) was prepared under simulated decoction conditions of SND. A mouse model of cardiomyopathy was established by intraperitoneal injection of Dox, followed by oral administration of SND, SAC, SMC, and GBMA, to evaluate improvements in cardiac systolic function across treatment groups. The effects of SND and its components on cardiac systolic function were evaluated to reveal the differences in their medicinal effects. The mechanisms by which SND and SAC improve cardiomyopathy were systematically investigated using Seahorse energy metabolism analysis, autophagic flux analysis, and Western blotting, with a focus on the interconnected organelles, mitochondria, and autophagosomes. RESULTS: A total of 173 compounds were identified in SAC. Among them, molecules with lower hydrophilicity tend to self-assemble in the decoction, while molecules with a molecular weight less than 300 Da participate less in self-assembly. SND, SAC, and GBMA significantly improved abnormal cardiac systolic function in Dox-induced cardiomyopathy mice, while SMC had no such effects. SAC and GBMA inhibited Dox-induced excessive mitophagy by downregulating BNIP3 and MFN2 expression, thereby protecting mitochondria and cardiomyocyte nuclei. SND, SAC, and GBMA all alleviated mitochondrial functional impairment in the cardiomyopathy model. They could trigger energy metabolism reprogramming (reducing the expression of the pyruvate dehydrogenase complex subunits PDC-E2 and PDC-E3, and increasing the expression of OXPHOS-CI and OXPHOS-CIV) to optimize mitochondrial energy supply efficiency. SMC could downregulate CII expression in OXPHOS and exert some mitochondrial protective effects, but failed to significantly improve mitochondrial functional impairment. CONCLUSIONS: SND and SAC administration improve Dox-induced cardiomyopathy. They could protect mitochondria and cardiomyocyte nuclei through optimizing mitochondrial energy metabolism efficiency and inhibiting autophagy, while the specific active constituents and their absorption routes remain to be determined. This study elucidates the molecular mechanism by which the adminstration of SAC in SND improves Dox-induced cardiomyopathy, providing new scientific evidence for the combined clinical application of SND and Dox. It also offers a new approach to the research of the substance basis of TCM prescriptions, namely, focusing on the self-assembled substances present in the decoction, rather than assuming the active components are small molecules, to accurately reveal the overall effects of TCM prescriptions and the scientific connotation of their compatibility. It should be noted that the current findings are based on a single batch of SND, and further validation using multiple batches is required to confirm the consistency of the observed effects.