Cardioprotective role of azafrin in against myocardial injury in rats via activation of the Nrf2-ARE pathway

Background: The root of Centranthera grandiflora Benth. is a Chinese ethnodrug known as Ye-Can-Dou-Gen (YCDG) and has been widely used to treat cardiovascular diseases in Hmong, Yunnan Province, China; however, its mechanisms of action were largely unknown. Azafrin, a natural carotenoid, is one of the most abundant active ingredients in YCDG. Purpose: We aimed to explore the cardioprotective effects of azafrin on myocardial infarction (MI) and myocardial ischaemia-reperfusion (MIR) injury and to clarify its underlying myocardial protective mechanisms. Methods: The in vitro cardioprotective effects of azafrin against oxidative stress injury in H9c2 cells were assessed by the ferric reducing antioxidant power (FRAP) assay and a viability assay in the presence of H2O2-induced oxidative insults, whereas the in vivo effects were assessed with the echocardiographic and histopathological evaluation together with 2,3,5-triphenyltetrazolium chloride (TTC) and serum biochemical analyses in vivo. Then, we used the luciferase reporter gene assay to measure Nrf2 transcriptional activity in HEK 293 cells and analysed the expression levels of enzymes downstream of Nrf2-ARE signalling by q-PCR and western blotting. Results: Azafrin showed higher FRAP values than FeSO4 and exhibited prophylactic and therapeutic potency against H2O2-induced cell injury. Additionally, azafrin treatment dramatically improved cardiac function; ameliorated the infarct size and pathological changes in rats; decreased the levels of MDA, cTnI, and myocardial enzymes and elevated SOD activity in vivo. In addition, azafrin significantly elevated the transcriptional activity of Nrf2 in HEK 293 cells and up-regulated the mRNA expression levels of genes downstream of Nrf2-ARE pathway activation in H9c2 cells. Furthermore, the protein expression levels of HO-1 and NQO1 in the cytoplasm and Nrf2 in the nucleus were also up-regulated in both the in vivo and in vitro experiments. Conclusion: Azafrin exhibited cardioprotective effects against myocardial injury via activation of the Nrf2-ARE pathway. As a natural carotenoid antioxidant, this molecule represents a promising cardioprotective adjuvant for the prophylaxis and treatment of ischaemic heart diseases.