Abstract: BACKGROUND: Clinical observations have shown that Zhigancao Decoction can effectively improve the symptoms and signs of doxorubicin-induced cardiotoxicity and protect cardiac function. Nevertheless, its exact mechanism of action remains unclear.
OBJECTIVE: To explore the protective effect and mechanism of Zhigancao Decoction on myocardial injury induced by doxorubicin. 
METHODS: (1) The targets of Zhigancao Decoction were obtained from PharmMapper, TCMSP, PubChem and BATMAN-TCM databases, and mitochondria-related genes were obtained from MitoCarta 3.0. Intersection genes were screened for myocardial injury-related targets by GEO database. (2) Cell experiment: Thirty Sprague-Dawley rats were given 12.15 g/kg/d Zhigancao Decoction by gavage (in two doses) for 3 consecutive days. Blood samples were collected from the abdominal aorta 2 hours after the last dose, and the serum was separated to obtain the drug-containing serum. Passage 10 rat cardiomyocytes (H9C2 cells) were divided into five groups: the control group without any intervention, the model group treated with adriamycin for 48 hours, the low, medium, and high dose drug-containing serum group treated with 5%, 10%, and 20% drug-containing serum for 6 hours, and then treated with adriamycin for 48 hours. At the end of the interventions, the levels of lactate dehydrogenase and creatine kinase in the cell supernatant were measured, and the protein expression of Caspase-7, cellular mitochondrial ultrastructure, cell apoptosis, reactive oxygen species level, and the localization and distribution of α-actinin were detected. (3) Animal experiment: Thirty C57BL/6 mice were randomly divided into five groups with six mice in each group: the control group was subcutaneously injected with saline, the model group and the low, medium and high dose Zhigancao Decoction groups were injected intraperitoneally with adriamycin (once a week for 4 weeks) to establish the myocardial injury model. Meanwhile, the low-, medium- and high-dose Zhigancao Decoction groups were given 8.775, 17.55 and 35.12 g/kg Zhigancao Decoction by gavage respectively (once a day for 4 weeks), and the control and model groups were given saline by gavage. After the final administration, left ventricular ejection fraction, serum lactate dehydrogenase and creatine kinase levels, structural changes in myocardial tissue, cardiomyocyte apoptosis and Caspase-7 protein expression in myocardial tissue were detected. 
RESULTS AND CONCLUSION: (1) Venn plots of Zhigancao Decoction targets, myocardial injury targets, and mitochondria-related genes showed that Caspase-7 was an intersection gene. (2) Cell animal: Compared with the model group, the levels of supernatant lactate dehydrogenase and creatine kinase, reactive oxygen species, apoptosis and the protein expression of Caspase-7 and α-actinin were significantly reduced in the medium- and high-dose drug-containing serum groups. Damage to the ultrastructure of cellular mitochondria was significantly reduced. (3) Animal experiment: Compared with the model group, serum lactate dehydrogenase and creatine kinase levels, cardiomyocyte apoptosis and Caspase-7 protein expression were significantly reduced, left ventricular ejection fraction increased, and myocardial tissue structure was significantly improved in the medium- and high-dose Zhigancao Decoction groups. To conclude, Zhigancao Decoction can prevent and treat doxorubicin-induced myocardial injury primarily to a certain extent by reducing oxidative stress and mitochondrial damage, inhibiting myocardial cell apoptosis, protecting cardiac function, and improving the damaged structure of myocardial tissue. In terms of inhibiting the endogenous apoptosis pathway, Caspase-7 may be a key target.

Key words: doxorubicin, myocardial injury, Zhigancao Decoction, cell apoptosis, oxidative stress, mitochondrial damage, engineered tissue construction