Sirt6通过调控P53/SLC7A11/GPX4通路抑制骨骼肌细胞铁死亡

Sirtuin 6 suppresses ferroptosis in skeletal muscle cells by regulating P53/SLC7A11/GPX4 pathway

目的:探讨骨骼肌细胞铁死亡的发生及sirtuin 6 (Sirt6)对其调控的分子机制。方法:将C2C12小鼠成肌细胞分为对照组、erastin(Era;铁死亡诱导剂)组、Era+ferrostatin-1(铁死亡拮抗剂)组、Era+MDL-800(Sirt6激动剂)组和Era+OSS-128167(Sirt6抑制剂)组;通过细胞活力和C2C12成肌细胞肌源性分化情况观察铁死亡诱导剂及拮抗剂的影响;RT-qPCR和Western blot测定成肌细胞及肌管中Sirt6、肌萎缩标志物、铁死亡标志物的mRNA及蛋白表达水平;进一步检测P53蛋白乙酰化水平、胞内亚铁离子(Fe2+)、活性氧(ROS)、谷胱甘肽(GSH)及脂质过氧化指标Liperfluo;免疫荧光法检测肌管分化标志物肌球蛋白重链(MHC)荧光信号。结果:Era降低成肌细胞活力和肌管分化质量,伴有Sirt6的mRNA和蛋白水平下降(P<0.05),肌萎缩标志物肌肉环指蛋白1(MuRF1)和肌萎缩F盒蛋白(MAFbx)表达增加(P<0.05)。激活Sirt6可抑制P53蛋白第381位赖氨酸乙酰化,降低P53表达水平,提高溶质载体家族7成员11(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)水平,改善ROS、GSH、Fe2+和脂质过氧化等铁死亡特异性指标(P<0.05),逆转铁死亡导致的肌肉负性变化。反之,抑制Sirt6将进一步加重铁死亡。结论:Sirt6可抑制P53蛋白乙酰化使其活性降低,通过调控P53/SLC7A11/GPX4信号通路抑制成肌细胞铁死亡,进而改善肌肉质量。

AIM:To investigate the occurrence of ferroptosis in skeletal muscle cells and its molecular mecha-nisms.METHODS:Mouse C2C12 myoblasts were divided into control group,erastin (Era;ferroptosis inducer) group,Era+ferrostatin-1 (ferroptosis antagonist) group,Era+MDL-800[sirtuin 6 (Sirt6) agonist]group,and Era+OSS-128167(Sirt6 inhibitor) group.The mRNA and protein expression levels of Sirt6,muscle atrophy markers and ferroptosis markers in C2C12 myoblasts or myotubes were determined by RT-qPCR and Western blot.Furthermore,acetylation levels of P53protein,intracellular ferrous ion (Fe2+),reactive oxygen species (ROS),glutathione (GSH) and lipid peroxidation index Liperfluo were examined.Immunofluorescence revealed the intensity of myosin heavy chain (MHC;a biomarker of myo-tube differentiation) fluorescence signal.RESULTS:Treatment with Era reduced the viability of C2C12 cells and the quality of differentiated C2C12 myotube,accompanied by decreased Sirt6 mRNA and protein levels (P<0.05).The mRNA and protein levels of muscle atrophy F-box protein (MAFbx) and muscle ring-finger protein 1 (MuRF1),markers of amyotrophy,were increased (P<0.05).Activation of Sirt6 inhibited acetylation of lysine 381 site in P53 protein,and decreased P53 expression.The Sirt6 agonist also increased solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression levels,and altered ferroptosis specific indicators such as ROS,GSH,Fe2+and lipid per-oxidation (P<0.05).Conversely,inhibition of Sirt6 promoted ferroptosis.CONCLUSION:Sirt6 inhibits the acetylation of P53 protein to reduce its activity,counteracts ferroptosis-associated cell death and improves muscle mass by regulating the P53/SLC7A11/GPX4 signaling pathway.