RESUMO
OBJECTIVE: MicroRNAs (miRNAs) have been shown to play an important role in myocardial ischemia/reperfusion (MI/R) injury. This study aimed to determine the role of miR-432 in MI/R injury. METHODS: We established a MI/R injury model by ligation/untying of the left anterior descending coronary artery, and used viral infection to regulate gene expression, such as that of miR-432 in vitro and in vivo, and used RT-qPCR to detect the expression of the gene at mRNA level. Finally, western blotting and immunochemistry analyses were used to determine the protein level. RESULTS: The results of this study show that miR-432 is upregulated in the heart following MI/R injury and that miR-432 overexpression showed a significant decrease, while miR-432 knockdown showed a significant increase in the ratio of the infarct area (IA) to the area at risk (AAR) and levels of serum creating phosphokinase (CPK). Moreover, miR-432 augmented the activation of the ß-catenin pathway and decreased the rate of apoptosis in the mice heart at 24 hours after MI/R injury by targeting RBM5. At the same time, miR-432 overexpression enhanced HIF-1α activation, while ß-catenin deletion attenuated HIF-1α activation induced by miR-432 overexpression. Importantly, ß-catenin and HIF-1α knockdown significantly increased the rate of apoptosis and the ratio of IA to AAR and levels of serum CPK induced by miR-432 overexpression at 24 hours after MI/R injury. miR-432 overexpression strongly decreased levels of SOD and GSH-PX activity, and increased levels of MDA activity and the expression of the gp91phox protein in the mice hearts at 24 hours after MI/R injury, while miR-432 knockdown exerted an opposite effect. miR-432 was also found to have increased NRF2 protein levels by targeting KEAP1 protein expression. NRF2 knockdown reversed the downregulation of the levels of gp91phox protein and MDA, while it also reversed the upregulation of the levels of SOD and GSH-PX induced by miR-432 overexpression in the heart of the mice at 24 hours after MI/R injury. CONCLUSION: miR-432 protects against MI/R injury by activating the ß-catenin/HIF-1α pathway and augmenting NRF2-mediated anti-oxidative stress.
RESUMO
Nesfatin-1 (encoded by NUCB2) is a cardiac peptide possessing protective activities against myocardial ischaemia/reperfusion (MI/R) injury. However, the regulation of NUCB2/nesfatin-1 and the molecular mechanisms underlying its roles in MI/R injury are not clear. Here, by investigating a mouse MI/R injury model developed with transient myocardial ischaemia followed by reperfusion, we found that the levels of NUCB2 transcript and nesfatin-1 amount in the heart were both decreased, suggesting a transcriptional repression of NUCB2/nesfatin-1 in response to MI/R injury. Moreover, cardiac nesfatin-1 restoration reduced infarct size, troponin T (cTnT) level and myocardial apoptosis, supporting its cardioprotection against MI/R injury in vivo. Mechanistically, the Akt/ERK pathway was activated, and in contrast, endoplasmic reticulum (ER) stress was attenuated by nesfatin-1 following MI/R injury. In an in vitro system, similar results were obtained in nesfatin-1-treated H9c2 cardiomyocytes with hypoxia/reoxygenation (H/R) injury. More importantly, the treatment of wortmannin, an inhibitor of Akt/ERK pathway, abrogated nesfatin-1 effects on attenuating ER stress and H/R injury in H9c2 cells. Furthermore, nesfatin-1-mediated protection against H/R injury also vanished in the presence of tunicamycin (TM), an ER stress inducer. Lastly, Akt/ERK inhibition reversed nesfatin-1 effects on mouse ER stress and MI/R injury in vivo. Taken together, these findings demonstrate that NUCB2/nesfatin-1 inhibits MI/R injury through attenuating ER stress, which relies on Akt/ERK pathway activation. Hence, our study provides a molecular basis for understanding how NUCB2/nesfatin-1 reduces MI/R injury.
