Carvedilol Protects Against the H₂O₂-induced Cell Damages in Rat Myoblasts by Regulating the Circ_NFIX/miR-125b-5p/TLR4 Signal Axis

Circular RNAs (circRNAs) have been involved in the regulation of various kinds of cardiovascular diseases, including acute myocardial infarction. This study was performed to investigate the molecular mechanism associated with circRNA nuclear factor IX (circ_NFIX) in carvedilol-mediated cardioprotection in H2O2-treated H9c2 cells. Flow cytometry was performed for the analysis of cell cycle and apoptosis. Cell proliferation was evaluated using colony formation assay and 3-(4,5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide assay. Lactate dehydrogenase (LDH) activity was measured by LDH assay. The relative levels of circ_NFIX, microRNA-125b-5p (miR-125b-5p), and toll-like receptor 4 (TLR4) were determined by quantitative real-time polymerase chain reaction. Protein levels were examined by Western blot. The target interaction was proved by dual-luciferase reporter assay. H2O2-induced cell cycle arrest, proliferation repression, apoptosis, and LDH promotion in H9c2 cells were inhibited by carvedilol. The circ_NFIX level was reduced after carvedilol treatment in H2O2-treated H9c2 cells, and circ_NFIX overexpression inhibited the protective effects of carvedilol on H2O2-induced cell damages. Furthermore, circ_NFIX was validated to serve as a sponge of miR-125b-5p, and the inhibitory function of circ_NFIX in carvedilol-induced cardioprotection was achieved by sponging miR-125b-5p. Moreover, TLR4 acted as a target gene of miR-125b-5p and miR-125b-5p inhibitor upregulated the TLR4 expression to suppress the protective effects of carvedilol on H2O2-treated H9c2 cells. In addition, circ_NFIX regulated the TLR4 level by exerting the sponge influence on miR-125b-5p. The rat model also indicated that Carv might suppress the progression of acute myocardial infarction by regulating the levels of circ_NFIX, miR-125b-5p, and TLR4. These findings suggested that carvedilol protected H9c2 cells against the H2O2-induced cell dysfunction through depending on the circ_NFIX/miR-125b-5p/TLR4 axis.