Astragalus membranaceus injection activates mitophagy and protects mitochondrial function in chronic heart failure...

To investigate the effects and mechanisms of Astragalus Membranaceus Injection (AMI) on mitophagy and mitochondrial function in chronic heart failure (CHF) based on phosphoproteomic and network pharmacology analysis. Primary neonatal mouse cardiomyocytes were isolated and hypertrophy cardiomyocyte model was induced by phenylephrine (PE) stimulation. AMI's effects on cell size, apoptosis, mitophagy, and mitochondrial function in hypertrophic cardiomyocytes were assessed. A pressure-overload CHF model was established via transverse aortic constriction (TAC) surgery in C57BL/6N mice. Echocardiography and histopathology were employed to evaluate AMI's effects on cardiac function and structural remodeling. Transmission electron microscope (TEM) and immunofluorescence were used to detect the distribution of autophagosomes and mitochondria. Phosphorylation-antibody microarray and network pharmacology were employed to explore AMI's cardioprotective mechanisms. The AKT/mTOR pathway's involvement was verified through Western blotting of AKT^Ser473 and mTOR^Ser2481 phosphorylation and pharmacological validation using SC79 (AKT/mTOR activator) and GSK-690693 (AKT/mTOR inhibitor) in gain/loss-of-function experiments. In vitro, AMI dose-dependently suppressed pathological hypertrophy, attenuated apoptosis, restored mitochondrial function, and enhanced mitophagic flux. In vivo, AMI treatment significantly improved left ventricular ejection fraction while attenuated cardiac hypertrophy and interstitial fibrosis in TAC-induced CHF mice. Besides, AMI treatment increased the number of mitochondria and elevated autophagy in TAC mice. Phosphoproteomic screening and network pharmacology analysis identified the PI3K/AKT/mTOR axis as the primary regulatory pathway mediating AMI's cardioprotection. Pharmacological activation of AKT/mTOR signaling using SC79 significantly suppressed mitophagic flux, whereas AMI treatment mirrored the effects of the AKT/mTOR inhibitor GSK-690693, effectively restoring mitophagy and mitochondrial homeostasis. AMI exerts its cardioprotective effects through inhibition of the AKT/mTOR pathway, thereby ameliorating maladaptive remodeling and mitochondrial dysfunction in CHF.