Zishen Huoxue Decoction alleviates myocardial ischemia-reperfusion injury through dysregulation of Endoplasmic reticulum-Mitochondria homeostasis mediated by DUSP1-NDUFS4

Background and objective: Zishen Huoxue Decoction can ameliorate myocardial ischaemia by regulating the mitochondrial quality control network. However, the identification of new molecular targets is necessary for ZSHX's control of mitochondrial protein homeostasis and metabolic activities. Methods: Animal and cellular models of NDUFS4^CKO or DUSP1^CKO were constructed, and the effects of ZSHX on endoplasmic reticulum stress and mitochondrial metabolism were analysed using a combination of single-cell sequencing, metabolomics, network pharmacology, and in vivo and in vitro drug intervention. Results: Endoplasmic reticulum stress and mitochondrial metabolic reprogramming were triggered by myocardial I/R damage, which also caused DUSP1/NDUFS4 downregulation. The role of ZSHX in controlling mitochondrial activity in response to myocardial inflammatory damage was tentatively confirmed by network pharmacology. Metabolomics results confirmed that the ZSHX intervention changed the composition and expression of metabolites in the I/R group. The association between myocardial I/R and mitochondrial energy metabolism and cell death was further tested by single-cell sequencing. The in vitro findings demonstrated that ZSHX protected mitochondrial proteostasis and normal biological functions, inhibited endoplasmic reticulum stress injury, restored calcium recycling/release balance, upregulated DUSP1/NDUFS4 expression, and controlled mitochondrial metabolic reprogramming to reduce myocardial inflammatory injury. The primary active component in the ZSHX formula is kaempferol. Conclusion: The primary active component of ZSHX formula, kaempferol, reduced myocardial I/R injury by upregulating the expression of DUSP1/NDUFS4, preventing endoplasmic reticulum stress and inflammatory burst, preserving mitochondrial function, and re-encoding mitochondrial metabolic processes following I/R injury.