HIIT对高脂喂养ApoE敲除鼠骨骼肌氧化应激的影响及肌肉因子BAIBA的保护作用

Effects of HIIT on Oxidative Stress in the Skeletal Muscle of Apo^(E-/-)Mice Fed with a High-fat Diet and the Protective Effects of BAIBA

目的:探讨高强度间歇训练(high-intensity interval training,HIIT)对高脂喂养的载脂蛋白E(ApoE)敲除(Apo^(E-/-))小鼠骨骼肌氧化应激的改善作用以及肌肉因子β-氨基异丁酸(β-aminoisobutyic acid,BAIBA)在其中的可能调节机制。方法:9周龄健康雄性C57BL/6J小鼠和Apo^(E-/-)小鼠分别喂食标准和高脂饲料,其中C57BL/6J小鼠为基线对照组(WT,n=6),Apo^(E-/-)小鼠经高脂饲料喂养12周后随机分为Apo^(E-/-)安静组(Apo^(E-/-),n=10)和Apo^(E-/-)高强度间歇训练组(Apo^(E-/-)HIIT,n=10),Apo^(E-/-)HIIT组进行6周HIIT,WT和Apo^(E-/-)组在笼中自由活动。最后一次运动后48 h取小鼠两侧腿部腓肠肌进行氧化损伤、抗氧化及BAIBA生成相关指标的检测。随后采用L-BAIBA预孵育C2C12细胞,后用过氧化氢(H_(2)O_(2))诱导氧化应激,结合干预条件随机分为Control、H_(2)O_(2)和L-BAIBA+H_(2)O_(2)组。相差显微镜观察细胞形态,并检测细胞ROS水平和Nrf2介导的抗氧化通路的相关指标。结果:1)与WT组相比,Apo^(E-/-)组小鼠骨骼肌氧化损伤相关的4-HNE修饰蛋白、蛋白质羰基含量和ROS水平显著升高,而抗氧化相关的p-Nrf2蛋白表达和GSH含量显著降低;而与Apo^(E-/-)组相比,Apo^(E-/-)HIIT组小鼠骨骼肌4-HNE修饰蛋白和蛋白质羰基含量明显降低,p-Nrf2蛋白表达和GSH含量明显升高。2)Apo^(E-/-)HIIT组小鼠骨骼肌生成BAIBA的相关酶基因Acads和Hadha mRNA的表达水平显著高于Apo^(E-/-)组。3)与Control组相比,H_(2)O_(2)干预后C2C12细胞明显皱缩,活细胞数量降低,ROS水平显著增加;而与H_(2)O_(2)组相比,L-BAIBA+H_(2)O_(2)组活细胞数量增加,形态较饱满,ROS水平明显降低。4)与Control组相比,H_(2)O_(2)显著降低了细胞Nrf2、p-Nrf2及其介导的抗氧化酶SOD1、NQO1和CAT蛋白以及合成GSH的相关酶基因Gss、Gsr和Gclm mRNA的表达水平,并显著降低了细胞GSH含量;而L-BAIBA+H_(2)O_(2)组较H_(2)O_(2)组则显著增加了p-Nrf2、SOD1和NQO1蛋白以及Gss、Gsr、Gclc和Gclm mRNA的表达量,同时细胞GSH含量也显著增加。结论:6周HIIT降低了高脂喂养的Apo^(E-/-)小鼠骨骼肌的氧化损伤,其中肌肉因子BAIBA对Nrf2介导的抗氧化信号的激活可能起着一定的作用。

Objective:To investigate the effects of high-intensity interval training(HIIT)on oxidative stress in the skeletal muscle of Apo^(E-/-)mice fed with a high-fat diet,and the possible regulatory mechanism ofβ-aminoisobutyric acid(BAIBA)in these effects.Methods:9-week-old healthy male C57BL/6J and Apo^(E-/-)mice were fed a standard or high-fat diet,respectively.C57BL/6J mice were the baseline control group(WT,n=6),and Apo^(E-/-)mice were randomly assigned to sedentary(Apo^(E-/-),n=10)and HIIT(Apo^(E-/-)HIIT,n=10)groups after 12 weeks of high-fat feeding.The Apo^(E-/-)HIT group received 6 weeks of HIT,WT and Apo^(E-/-)groups received no intervention.Forty-eight hours after the last exercise session,the whole gastrocnemius muscle of both hind limbs of the mice were taken for the detection of oxidative damage,antioxidant,and BAIBA production-related indicators.In vitro experiment,C2C12 cells were pre-incubated with L-BAIBA,and then oxidative stress was induced with hydrogen peroxide(H_(2)O_(2)).Combined with the intervention,they were randomly divided into Control,H_(2)O_(2)and LBAIBA+H_(2)O_(2)groups,then the cell morphology,ROS level and related indicators of Nrf2-mediated antioxidant pathways were determined.Results:1)Compared with the WT group,Apo^(E-/-)mice increased the expression of 4-HNE modified protein,protein carbonylation content and ROS level,while decreased the protein expression of p-Nrf2 and the concentration of CSH in the skeletal muscle.Compared with the Apo^(E-/-)group,HIT significantly decreased the expression of 4-HNE modified protein,protein carbonylation content,while increased the protein expression of p-Nrf2 and the concentration of CSH.2)The mRNA expression of Acads and Hadha related to the production of BAIBA in the skeletal muscle of mice in the Apo^(E-/-)HIT group were significantly higher than those in the Apo^(E-/-)group.3)Compared with the Control group,C2C12 cells in the H_(2)O_(2)group shrank significantly,the number of viable cells decreased,and the level of ROS increased under the stimulation of H_(2)O_(2)oxidative stress.While the morphology,number and ROS level of viable cells were significantly improved in the L-BAIBA+H,O2 group compared with the H,O2 group.4)Compared with the Control group,the protein expression of Nrf2,p-Nrf2 and their mediated antioxidant enzymes SOD1,NQO1,CAT,as well as the mRNA expression of Gss,Gsr,Gclm related to GSH synthesis and the concentration of GSH in the H_(2)O_(2)group were significantly decreased.While LBAIBA preincubation increased the protein expression of p-Nrf2,SOD1,NQO1 and the mRNA expression of Gss、Gsr、Gclc,Gclm,as well as the concentration of CSH in the C2C12 cells under the stimulation of H_(2)O_(2).Conclusion:6-week HIIT can ameliorate oxidative damage in the skeletal muscle of Apo^(E-/-)mice fed with a high-fat diet,in which the activation of Nrf2-mediated antioxidant signaling by the myokine of BAIBA may play an important role.