Apelin-13对高铁环境中骨骼肌细胞C2C12细胞铁死亡的影响

Effects of apelin-13 on ferroptosis of the C2C12 skeletal muscle cell line in a high-iron environment

目的观察Apelin-13对高铁环境中骨骼肌细胞C2C12细胞铁死亡的影响并探讨其可能的机制。方法C2C12细胞培养在改良Eagle培养基(DMEM)中,实验分为对照组、柠檬酸铁胺(FAC)组、Apelin-13组、Apelin-13+FAC组、铁死亡诱导剂RSL3组和Apelin-13+FAC+RSL3组。细胞活力采用二苯基四氮唑溴盐(MTT)法检测;采用比色法检测细胞内总铁离子和二价铁离子浓度,酶联免疫法检测细胞中谷胱甘肽(GSH)水平,可见分光光度法检测细胞中丙二醛(MDA)水平,化学荧光法检测细胞内活性氧(ROS)水平;透射电镜检测C2C12细胞超微结构。蛋白质印迹分析检测谷胱甘肽过氧化物酶4(GPX-4)、铁蛋白重链-1(FTH-1)、血红素加氧酶(HO)-1和核因子E2相关因子-2(Nrf-2)蛋白的表达水平。结果与FAC组比较,FAC+Apelin-13组细胞活力显著增加(光密度值0.52±0.06比0.28±0.04,t=7.837、P=0.007),细胞中GSH的浓度显著增加[(2.41±0.35)μmol/g Pro比(0.91±0.12)μmol/g Pro,t=9.778,P=0.003],细胞中ROS[(22.06±5.79)a.u./mg Pro比(52.71±7.28)a.u./mg Pro,t=8.064,P=0.006]、MDA[(4.63±0.51)mmol/mg Pro比(9.11±0.84)mmol/mg Pro,t=8.642,P=0.006]、总铁离子[(1.53±0.24)μmol/g Pro比(3.17±0.55)μmol/g Pro,t=6.135、P=0.013]和二价铁离子[(0.75±0.08)μmol/g Pro比(1.94±0.36)μmol/g Pro,t=5.068、P=0.027]的水平降低,细胞内铁沉积明显减少。对照组和Apelin-13组线粒体结构清晰,形态正常;FAC组细胞内线粒体出现膜密度增加,膜皱缩及破裂,线粒体内部存在空泡变性,出现明显线粒体损伤,符合铁死亡的形态学特征;与FAC组比较,FAC+Apelin-13组内线粒体损伤情况明显改善。与FAC+Apelin-13组比较,FAC+Apelin-13+RSL3组细胞活力显著降低(光密度值0.23±0.04比0.48±0.06,t=7.642、P=0.007)。与FAC组比较,FAC+Apelin-13组细胞中GPX-4(相对表达水平0.96±0.14比0.31±0.07,t=7.712、P=0.008)和FTH-1(相对表达水平0.57±0.08比0.27±0.05,t=6.944、P=0.011)蛋白的表达水平显著上调,Nrf-2在C2C12细胞核中的表达(相对表达水平0.42±0.04比0.19±0.05,t=7.114、P=0.008)及Nrf-2在细胞核中表达与Nrf-2总蛋白表达水平的百分比[(58.36±5.24)%比(36.58±5.32)%,t=5.858、P=0.015]以及HO-1的蛋白表达水平(相对表达水平0.49±0.07比0.28±0.05,t=6.472、P=0.012)均显著升高。结论Apelin-13抑制了高铁环境诱导的C2C12细胞铁死亡,其机制可能涉及到Nrf-2/HO-1信号通路。

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±0.12μmol/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±0.55μmol/g Pro,t=6.135,P=0.013)and divalent iron(0.75±0.08 vs.1.94±0.36μmol/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.