Effects of apelin-13 on ferroptosis of the C2C12 skeletal muscle cell line in a high-iron environment / 中华老年医学杂志

ABSTRACT

Objective:To examine the effects of apelin-13 on ferroptosis of the C2C12 skeletal muscle cell line induced by a high-iron environment and explore potential underlying mechanisms.Methods:C2C12 cells were cultured in Dulbecco's Modified Eagle Medium(DMEM)and divided into a control group, a ferric citrate(FAC)group, an apelin-13 group, an FAC+ apelin-13 group, a ferroptosis inducer RSL3 group and an FAC+ apelin-13+ RSL3 group.Cell viability was detected by the 3-(4, 5-dimethyl thiazole-2)-2, 5-diphenyl thiazolyl blue(MTT)assay.The intracellular concentrations of total iron and divalent iron were measured by colorimetry; the levels of glutathione(GSH), malondialdehyde(MDA)and intracellular reactive oxygen species(ROS)in cells were detected by an enzyme-linked immunosorbent assay, visible spectrophotometry and a chemifluorescence method, respectively.The ultrastructure of C2C12 cells was examined by transmission electron microscopy.The protein expression of glutathione peroxidase 4(GPX-4), ferritin heavy chain 1(FTH-1), heme oxygenase 1(HO-1)and nuclear factor E2-related factor 2(Nrf-2), were detected by Western blotting.Results:Compared with the FAC group, the FAC+ Apelin-13 group had significantly elevated cell viability(optical density 0.52±0.06 vs.0.28±0.04, t=7.837, P=0.007)and higher concentrations of GSH(2.41±0.35 vs.0.91±0.12 μmol/g Pro, t=9.778, P=0.003), but significantly decreased levels of ROS(22.06±5.79 vs.52.71±7.28 a. u./mg Pro, t=8.064, P=0.006), MDA(4.63±0.51 vs.9.11±0.84 mmol/mg Pro, t=8.642, P=0.006), total iron(1.53±0.24 vs.3.17±0.55 μmol/g Pro, t=6.135, P=0.013)and divalent iron(0.75±0.08 vs.1.94±0.36 μmol/g Pro, t=5.068, P=0.027), as well as reduced intracellular iron deposition.In the control group and the apelin-13 group, the morphology of the mitochondria was clear and normal.In contrast, the mitochondria in the FAC group had increased membrane density, membrane shrinkage and rupture, vacuolar degeneration, and obvious mitochondrial damage, which were consistent with the morphological characteristics of ferroptosis.Compared with the FAC group, the FAC+ apelin-13 group showed significant improvement in mitochondrial damage.Moreover, compared with the FAC+ apelin-13 group, the cell viability of the FAC+ apelin-13+ RSL3 group was significantly decreased(optical density 0.23±0.04 vs.0.48±0.06, t=7.642, P=0.007). Compared with the FAC group, the FAC+ apelin-13 group had significantly up-regulated cellular expression of GPX-4(relative expression 0.96±0.14 vs.0.31±0.07, t=7.712, P=0.008), FTH-1(0.57±0.08 vs.0.27±0.05, t=6.944, P=0.011), and HO-1(0.49±0.07 vs.0.28±0.05, t=6.472, P=0.012), as well as increased nuclear expression of Nrf-2(relative expression 0.42±0.04 vs.0.19±0.05, t=7.114, P=0.008)with a higher ratio of nuclear expression over total cellular expression[(58.36±5.24)% vs.(36.58±5.32)%, t=5.858, P=0.015]and a higher level of HO-1 protein expression(relative expression 0.49±0.07 vs.0.28±0.05, t=6.472, P=0.012). Conclusions:Apelin-13 inhibits ferroptosis induced by a high iron environment in C2C12 cells, and the underlying molecular mechanisms may be related to the Nrf-2/HO-1 signaling pathway.