摘要目的 观察高铁培养环境下小鼠成肌细胞C2C12生物学活性指标的变化,探讨高铁应激对骨骼肌细胞生物学活性的影响和相关机制.方法 体外培养小鼠成肌细胞C2C12,在马血清诱导作用下分化为骨骼肌细胞,以不同浓度(50、100、200 μmol/L)枸橼酸铁铵(FAC)干预,细胞计数试剂盒(CCK-8)检测细胞增殖,流式细胞仪检测细胞凋亡率,活性氧(ROS)检测试剂盒检测细胞内ROS水平,线粒体荧光探针标记线粒体,流式细胞仪检测细胞内线粒体相对含量,Western blot法检测细胞内自噬相关蛋白轻链蛋白3Ⅰ、Ⅱ(LC3 Ⅰ、LC3Ⅱ)、B淋巴细胞瘤2/腺病毒相互作用蛋白3及其同族体(BNIP3、BNIP3L)的表达;Western blot法分别检测细胞色素C(Cyt C)在细胞质和线粒体内的表达,使用抗氧化剂N-乙酰半胱氨酸(NAC 2.5 mmol/L)和FAC共同干预,再次检测细胞的增殖、凋亡指标.结果 C2C12细胞在FAC干预后,细胞的增殖活性随FAC干预浓度增加呈浓度依赖性下降(P＜0.01),凋亡率呈浓度依赖性升高(P＜0.01);ROS水平呈浓度依赖性升高(P＜0.01);线粒体含量呈浓度依赖性升高(P＜0.01);LC3Ⅱ/LC3 Ⅰ、BNIP3、BNIP3L蛋白的表达呈浓度依赖性增加(P＜0.01);Cyt C胞质/线粒体蛋白的表达呈浓度依赖性增加(P＜0.01);加入NAC后,FAC+ NAC组、FAC组细胞增殖活性终值分别为1.46 ±0.05、0.63 ±0.04;凋亡率分别为4.72±0.57、15.78±0.57,两组比较均有统计学意义(P＜0.01).结论 高铁应激环境可抑制骨骼肌细胞的活性,其机制可能与高铁介导的氧化水平增加并进一步激活线粒体自噬和凋亡有关.

abstractsObjective To investigate the effects of stress caused by iron excess on biologic activity of murine myoblast C2C12 cells and related mechanism.Methods Murine preosteoblast myoblast C2C12 cells were incubated in a medium supplemented with different concentrations (50,100,200 μmol/L) of ferric ammonium citrate (FAC) under induction of horse serum.The proliferation were assessed by cell counting kit (CCK-8).The apoptotic hallmarks were detected by flow cytometer.Intracellular reactive oxygen species (ROS) was measured using dichlorodihydrofluorescein diacetate (DCFH-DA).The quantity of intracellular mitochondria was measured using mitochondrial assay kit.The expression of autophagy proteins microtubule-associated protein 1 light 3Ⅰ (LC3Ⅰ),LC3Ⅱ,bcl-2/adenovirus E1B 19-kilodalton interacting protein3 (BNIP3),and bcl-2/adenovirus E1B 19-kilodalton interacting protein3-like (BNIP3L)were detected by Western blotting.The expression of cytochrome C in the cytoplasm and mitochondria were detected by Western blotting respectively.The cells were treated with 2.5 mmoL/L antioxidant N-acetyl cysteine (NAC) and FAC,the proliferation and apoptosis indexes of the cells were tested again.Results Proliferation of C2C12 cells was significantly inhibited by FAC in dose dependent manner (P ＜ 0.01).The apoptotic rate,level of ROS,and number of mitochondria were increased by FAC in dose dependent manner (P ＜0.01).The protein expression of LC3 Ⅱ/LC3 Ⅰ,BNIP3,and BNIP3L were increased by FAC in dose dependent manner (P ＜ 0.01).The protein expression of Cyt C cytoplasm/mitochondria increased by FAC in dose dependent manner (P ＜ 0.01).The proliferation indices in FAC + NAC,FAC groups were 1.46 ±0.05 and 0.63 ±0.04 respectively,and apoptotic rate were 4.72 ±0.57 and 15.78 ±0.57 respectively.The statistically significant differrence (P ＜ 0.01) suggested that addition of NAC partly reversed the decrease of proliferation and the increase of apoptosis in C2C12 cells induced by FAC.Conclusion The stress caused by iron excess can significantly inhibit biologic activity of skeletal muscle cells,and the mechanism behind inhibition may involve activation of autophagy and apoptosis of mitochondria induced by oxidative stress.