Circ_0000003 Prevents H2O2-Induced H9C2 Cells Injury by Inhibiting the TLR4/NF-κB p65 Pathway

Abstract

Background: Circular RNA (circRNA) plays a crucial role in several cerebral cardiovascular disorders; however, the mechanism of coronary heart disease (CHD) is still not fully understood. Circ_0000003 is one of the circRNAs which affects the cell proliferation or apoptosis and may have a regulatory function in CHD. Thus, the aim of this study is to explore the role of circ_0000003 in CHD and its mechanism. Methods: The blood samples of clinical CHD patients (CHD group, n = 20) and healthy volunteers (normal group, n = 20) were collected. The expression level of circ_0000003 in the blood was observed by quantitative real-time polymerase chain reaction (qRT-PCR). To mimic CHD in vitro, H9C2 rat cardiomyocytes were induced with 50 μmol/L H₂O₂, and the expression levels of circ_0000003 in the resulting cardiomyocytes were recorded. The circ_0000003 overexpression vector (circ_0000003) and negative control (vector), as well as the shRNA circ_0000003 vector (sh-circ_0000003) and shRNA negative control (sh-NC) were transfected into H₂O₂-induced H9C2 cells. Cell counting kit-8 (CCK-8) assay and propidium iodide (PI)/fluorescein isothiocyanate (FITC)-Annexin V staining were performed to determine the cell proliferation and apoptosis levels, as well as the levels of inflammation-related factors interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α and oxidative stress-related factors malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) in each group. The expression levels of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) p65 pathway-related proteins (TLR4, phosphorylation-nuclear factor kappa-B (p-NF-κB), NF-κB) in each group of cells were observed by western blotting. Results: The expression levels of circ_0000003 were significantly reduced in CHD patients and CHD cell models (p < 0.01). When circ_0000003 was overexpressed in the CHD cell model, it greatly promoted cell proliferation and inhibited apoptosis (p < 0.01). On the other hand, the overexpression decreased the levels of IL-6, IL-1β, TNF-α, MDA, and ROS, but increased SOD levels (p < 0.01). In addition, TLR4 expression and p-NF-κB/NF-κB ratio were lowered by circ_0000003 overexpression in the CHD cell model (p < 0.01). More importantly, the knockdown of circ_0000003 showed the opposite results to those by overexpression of circ_0000003. Conclusions: Circ_0000003 inhibits H₂O₂-induced inflammatory and oxidative stress responses in H9C2 cells, thereby promoting cell proliferation and inhibiting apoptosis, which is associated with blocking the TLR4/NF-κB p65 pathway.