Luhong Formula and Hydroxysafflor yellow A protect cardiomyocytes by inhibiting autophagy

Heart failure (HF) is the terminal stage of all heart diseases that is characterized by irreversible cardiomyocyte injury. Equilibrium of autophagy is essential for cardiac cell survival. The Luhong formula (LHF) has been clinically applied for decades, and has exhibited significant efficacy in improving heart function and alleviating the symptoms of angina pectoris.To clarify the mechanism of action of LHF and one of its main constituents, hydroxysafflor yellow A (HYSA), in protecting ischemic cardiomyocytes by inhibiting autophagy.Cell viability was detected by CCK-8 assay with LHF or HYSA pretreatment followed by hypoxic damage. Immunofluorescence of GFP-LC3-H9C2 and GFP-LC3-HeLa cells was used to observe autophagic flux. Beclin 1 and HIF1α protein expression were assessed using western blotting. LHF was orally administered to Wistar rats following myocardial infarcion. Echocardiography was performed before the rats were sacrificed; immunohistochemistry and western blotting were used to evaluate Beclin 1 and HIF1α expression in the myocardial tissue. Hematoxylin and eosin staining as well as Masson's trichrome staining were used to measure cardiac structure and myocardial fibrosis.LHF and HYSA reversed the hypoxia-induced decrease in cell viability in vitro. LHF and HYSA induced the aggregation of GFP-LC3 puncta and reduced the expression of Beclin 1 protein in H9C2, suggesting that LHF and HYSA may inhibit autophagy activity. Pretreatment with reactive oxygen species (ROS) inducers and inhibitors revealed that LHF and HYSA inhibited autophagy by suppressing cellular ROS. Further studies demonstrated that LHF and HYSA reduced the ROS levels by inhibiting HIF1α. LHF delayed fibrosis and protected heart function in vivo in a rat model of HF following myocardial infarction. Western blotting and immunohistochemistry revealed that LHF effectively reduced the expression of Beclin 1 and HIF1α in the infarcted area of the rat heart.These results demonstrate that hydroxysafflor yellow A is the representative bioactive compounent of Luhong Formula on regulating autophagy to protectect cardiomyocytes from hypoxia injury. LHF and HYSA inhibit cardiac autophagy by suppressing HIF1α-mediated ROS production. This study helps to further clarify the underlying mechanism of LHF and provide a scientific basis for its development as a novel cardiovascular therapeutic agent.