Bellidifolin from Gentianella acuta (Michx.) Hulten protects H9c2 cells from hydrogen peroxide-induced injury via the PI3K-Akt signal pathway

Cardiovascular Disease is the most common disease in the world and the first among the causes of human death. Its morbidity and mortality increase annually, but no effective treatment is available. Therefore, new drugs should be developed to treat Cardiovascular Disease. Gentianella acuta (Michx.) Hulten (G. acuta) is an important Mongolian medicine in China and elicits protective effects on cardiovascular health. In this study, liquid chromatography-mass spectrometry (LC-MS) combined with network pharmacology was used to screen the main active ingredients and confirm that bellidifolin was one of the main components for the treatment of ischemic heart disease. Then, rat myocardial (H9c2) cells injury model induced by hydrogen peroxide (H₂O₂) in vitro was established to verify the effect of bellidifolin on oxidative stress stimulation, including determination of antioxidant enzyme activity and Apoptosis. Transcriptome Sequencing, qRT-PCR, and western blot were performed to further verify the antioxidant stress mechanism of bellidifolin. Results showed that bellidifolin pretreatment decreased the rate of Apoptosis and the levels of Lactate Dehydrogenase (LDH), Creatine Kinase (CK), and alanine aminotransferase (ALT). Conversely, it increased the contents of superoxide dismutase (SOD) and Glutathione Peroxidase (GSH-Px) in a dose-dependent manner, indicating that bellidifolin caused a protective effect on cardiomyocyte injury. Bellidifolin minimized the H₂O₂-induced cell injury by activating the PI3K-Akt signal pathway and downregulating glycogen synthase kinase-3β (GSK-3β) and p-Akt1/Akt1. Therefore, this work revealed that G. acuta has a good development prospect as an edible medicinal plant in Cardiovascular Disease. Its bellidifolin component is a potential therapeutic agent for Cardiovascular Disease induced by oxidative stress damage.

ALT, alanine aminotransferase; AMPK, AMP-activated protein kinase; Akt1, protein kinase B1; BP, biological processes; Bellidifolin; CC, cellular components; CK, creatine kinase; DEGs, differentially expressed genes; G. acuta, Gentianella acuta(Michx.) Hulten; GO, gene ontology; GSH-Px, glutathione peroxidase; GSK-3β, glycogen synthase kinase-3β; H2O2-induced injury; H9c2, rat myocardial; IL-6, interleukin 6; KEGG, kyoto encyclopedia of genes and genomes; LDH, lactate dehydrogenase; Liquid chromatography-mass spectrometry; MF, molecular functions; Network pharmacology; Oxidative stress; PDB, protein data bank; PPI, protein-protein interaction; Protective effect; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF, tumor necrosis factor; TP53, tumor protein P53; VEGFA, vascular endothelial growth factor A; cAMP, cyclic adenosine monophosphate; qRT-PCR, quantitative reverse transcription PCR.