OBJECTIVE: To investigate the mechanism of Dan Ze mixture (丹泽合剂, DZM) in the treatment of lipotoxic cardiomyopathy.
METHODS: Ultra-performance liquid chromatography tandem mass spectrometry was employed to characterize the serum migration constituents of DZM. A lipotoxic cardiomyopathy rat model was established through high-fat diet and intervened by different doses of DZM. The cardiac function was assessed using echocardiography, and hematoxylin and eosin, oil red O, and Masson staining were conducted to evaluate morphological changes, lipid accumulation, and fibrosis in myocardial tissue. Serum myocardial enzyme activity, lipid levels, and lipid content of myocardial tissue were measured, while fluorescent staining and colorimetry were used to assess oxidation levels in myocardial tissue. Mitochondrial membrane potential was detected by 5,5’, 6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanineio-dide (JC-1). Transmission electron microscopy was employed to observe ultrastructure and mitochondrial structure changes in myocardial tissue. Fluorescence double staining and colocalization were utilized to observe the binding of autophagosomes and mitochondria, while immunohistochemical staining was used to detect the expression of mitophagy-related proteins. Terminal deoxynucleoitidyl transferase mediated nick end labeling staining was employed for the identification of apoptosis in myocardial tissue, while quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot were utilized for the detection of apoptosis, B-cell lymphoma-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3)/ mitophagy signaling pathway-related genes and proteins. In palmitic acid-induced Rat H9C2 cardiomyocytes (H9c2) cells, various cellular parameters including cell viability, lactate dehydrogenase release, apoptosis rate, oxidative stress level, mitochondrial structure and function, and mitophagy level were assessed after the treatment of DZM drug-containing serum for a duration of 24 h. The cellular expressions of BNIP3/mitophagy signaling pathway relevant genes and proteins were further evaluated using qRT-PCR and Western blot techniques.
RESULTS: A total of 295 prototypes (e.g., phenolic acids, quinones, terpenoids) were identified in serum of rats after oral administration of DZM. In vivo, DZM therapy has been shown to effectively enhance cardiac function, mitigate high-fat diet-induced myocardial structural damage and lipid accumulation. Furthermore, DZM has demonstrated the ability to reduce lipid levels, attenuate cell apoptosis, combat oxidative stress, enhance mitochondrial structure and function, and activate the BNIP3/mitophagy signaling pathway. Furthermore, the silencing of BNIP3 has been shown to exacerbate palmitic acid-induced damages in H9c2 cells, while inhibiting the BNIP3/mitophagy signaling pathway can mitigate the inhibitory effects of DZM on palmitic acid-induced apoptosis, lipid deposition and oxidative stress.
CONCLUSION: This study presents preliminary evidence for the therapeutic efficacy of DZM on lipotoxic cardiomyopathy through the activating BNIP3/mitophagy signaling pathway.